U.S. patent application number 17/148813 was filed with the patent office on 2021-07-15 for inhibitors of cancer invasion, attachment, and/or metastasis.
The applicant listed for this patent is SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE, VIRGINIA COMMONWEALTH UNIVERSITY. Invention is credited to Swadesh K. DAS, Surya K. DE, Luni EMDAD, Paul B. FISHER, Timothy P. KEGELMAN, Mitchell E. MENEZES, Maurizio PELLECCHIA, Jun WEI, Bainan WU.
Application Number | 20210214365 17/148813 |
Document ID | / |
Family ID | 1000005492748 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210214365 |
Kind Code |
A1 |
FISHER; Paul B. ; et
al. |
July 15, 2021 |
INHIBITORS OF CANCER INVASION, ATTACHMENT, AND/OR METASTASIS
Abstract
Provided herein are, inter alia, compositions that bind to a
PDZ1 domain of MDA-9/Syntenin (syndecan binding protein: SDCBP),
thereby inhibiting MDA-9/Syntenin activity, and methods of use of
same. The compositions and methods provided herein are useful for
treating cancer and preventing cancer metastasis, particularly in
cancers that have increased MDA-9/Syntenin expression.
Inventors: |
FISHER; Paul B.; (Henrico,
VA) ; PELLECCHIA; Maurizio; (Riverside, CA) ;
DAS; Swadesh K.; (Richmond, VA) ; KEGELMAN; Timothy
P.; (Richmond, VA) ; WU; Bainan; (Richmond,
VA) ; DE; Surya K.; (Richmond, VA) ; WEI;
Jun; (La Jolla, CA) ; MENEZES; Mitchell E.;
(Richmond, VA) ; EMDAD; Luni; (Richmond,
VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIRGINIA COMMONWEALTH UNIVERSITY
SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE |
Richmond
La Jolla |
VA
CA |
US
US |
|
|
Family ID: |
1000005492748 |
Appl. No.: |
17/148813 |
Filed: |
January 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16349467 |
May 13, 2019 |
11008325 |
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PCT/US2017/061443 |
Nov 14, 2017 |
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17148813 |
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62424571 |
Nov 21, 2016 |
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62421468 |
Nov 14, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 495/14 20130101;
C07D 487/04 20130101; A61K 31/519 20130101; A61P 35/04 20180101;
A61K 45/06 20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61P 35/04 20060101 A61P035/04; A61K 31/519 20060101
A61K031/519; A61K 45/06 20060101 A61K045/06; C07D 495/14 20060101
C07D495/14 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with government support under grant
nos. CA097318, CA 168517, and CA016059, awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A compound, or pharmaceutically acceptable salt thereof, having
the formula: ##STR00080## wherein R.sup.1 is independently halogen,
--CX.sup.1.sub.3, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
--OCX.sup.1.sub.3, --OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN,
--SO.sub.n1R.sup.1D, --SO.sub.v1NR.sup.1AR.sup.1B,
--NHC(O)NR.sup.1AR.sup.1B, --N(O).sub.m1, --NR.sup.1AR.sup.1B,
--C(O)R.sup.1C, --C(O) --OR.sup.1C, --C(O)NR.sup.1AR.sup.1B,
--OR.sup.1D, --NR.sup.1ASO.sub.2R.sup.1D, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)OR.sup.1C, --NR.sup.1AOR.sup.1C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.1
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.2 is independently hydrogen,
halogen, --CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--SO.sub.n2R.sup.2D, --SO.sub.v2NR.sup.2AR.sup.2B,
--NHC(O)NR.sup.2AR.sup.2B, --N(O).sub.m2, --NR.sup.2AR.sup.2B,
--C(O)R.sup.2C, --C(O)--OR.sup.2C, --C(O)NR.sup.2AR.sup.2B,
--OR.sup.2D, --NR.sup.2ASO.sub.2R.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O)OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; L.sup.1 is a bond,
--S(O).sub.2--, --N(R.sup.3)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--, --N(R.sup.3)C(O)NH--,
--NHC(O)N(R.sup.3)--, --S(O).sub.2N(R.sup.3)--,
--N(R.sup.3)S(O).sub.2--, --C(O)S(O).sub.2N(R.sup.3)--,
--N(R.sup.3)S(O).sub.2C(O)--, --C(O)O--, --OC(O)--, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, or substituted or unsubstituted
heteroarylene; R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --C(O)R.sup.3C,
--C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; L.sup.2is a bond,
--S(O).sub.2--, --N(R.sup.4)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--, --N(R.sup.4)C(O)NH--,
--NHC(O)N(R.sup.4)--, --S(O).sub.2N(R.sup.4)--,
--N(R.sup.4)S(O).sub.2--, --C(O)S(O).sub.2N(R.sup.4)--,
--N(R.sup.4)S(O).sub.2C(O)--, --C(O)O--, --OC(O)--, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, or substituted or unsubstituted
heteroarylene; R.sup.4 is independently hydrogen, --CX.sup.4.sub.3,
--CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN, --C(O)R.sup.4C,
--C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.1A, R.sup.1B,
R.sup.1C, R.sup.1D, R.sup.2A, R.sup.2B, R.sup.2C, R.sup.2D,
R.sup.3A, R.sup.3B, R.sup.3C, R.sup.4A, R.sup.4B, and R.sup.4C are
independently hydrogen, --CX.sub.3, --CN, --COOH, --CONH.sub.2,
--CHX.sub.2, --CH.sub.2X, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.1A and R.sup.1B substituents
bonded to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; R.sup.2A and R.sup.2B substituents bonded
to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; R.sup.3A and R.sup.3B substituents bonded
to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; R.sup.4A and R.sup.4B substituents bonded
to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; X, X.sup.1, X.sup.2, X.sup.3, and X.sup.4
are independently --F, --Cl, --Br, or --I; n1 and n2 are
independently an integer from 0 to 4; m1, m2, v1, and v2 are
independently 1 or 2; and z1 is an integer from 0 to 4; wherein
L.sup.2-R.sup.2 is not hydrogen, --OCH.sub.3, --CH.sub.3, or --Cl;
wherein R.sup.2 is not ##STR00081## R.sup.23 is independently
halogen, --CX.sup.23.sub.3, --CHX.sup.23.sub.2, --CH.sub.2X.sup.23,
--OCX.sup.23.sub.3, --OCH.sub.2X.sup.23, --OCHX.sup.23.sub.2, --CN,
--SO.sub.n23R.sup.100D, --SO.sub.v23NR.sup.100AR.sup.100B,
--NHC(O)NR.sup.100AR.sup.100B, --N(O).sub.m23, --N
R.sup.100AR.sup.100B, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --NR.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cvcloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl; two adjacent
R.sup.23 substituents may optionally be joined to form a
substituted or unsubstituted cvcloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.100A, R.sup.100B,
R.sup.100C, and R.sup.100D are independently hydrogen, --CX.sub.3,
--CN, --COOH, --CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cvcloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.100A and
R.sup.100B substituents bonded to the same nitrogen atom may
optionally be joined to form substituted or unsubstituted
heterocycloalkyl or substituted or unsubstituted heteroaryl;
X.sup.23 is independently --F, --Cl, --Br, or --I; n23 is
independently an integer from 0 to 4; m23, and v23 are
independently 1 or 2; and z23 is an integer from 0 to 2.
2. The compound of claim 1, wherein R.sup.1 is independently
halogen, --CX.sup.1.sub.3, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
--CH.sub.2X.sup.1, --OCX.sup.1.sub.3, --OR.sup.1D, --CN,
--NR.sup.1AR.sup.1B, substituted or unsubstituted alkyl or two
adjacent R.sup.1 substituents may optionally be joined to form a
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
3. (canceled)
4. The compound of claim 1, wherein R.sup.1 is independently
halogen, --OR.sup.1D, or --CH.sub.3.
5. (canceled)
6. (canceled)
7. The compound of claim 1, wherein L.sup.1 is a bond,
--S(O).sub.2--, --N(R.sup.3)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--, --N(R.sup.3)C(O)NH--,
substituted or unsubstituted alkylene, or substituted or
unsubstituted heteroalkylene.
8. (canceled)
9. (canceled)
10. (canceled)
11. The compound of claim 1, wherein L.sup.1 is --C(O)NH--.
12. The compound of claim 1, wherein L.sup.2 is a bond,
--S(O).sub.2--, --N(R.sup.4)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--, --N(R.sup.4)C(O)NH--,
substituted or unsubstituted alkylene, or substituted or
unsubstituted heteroalkylene.
13. (canceled)
14. (canceled)
15. (canceled)
16. The compound of claim 1, wherein L.sup.2 is substituted 2 to 6
membered heteroalkylene.
17. The compound of claim 1, wherein L.sup.2is
--NHC(O)CH.sub.2CH.sub.2--.
18. The compound of claim 1, wherein R.sup.2 is independently
hydrogen, halogen, --CX.sup.2.sub.3, --CHX.sup.2.sub.2,
--CH.sub.2X.sup.2, --OCX.sup.2.sub.3, --OCH.sub.2X.sup.2,
--OCHX.sup.2.sub.2, --NR.sup.2AR.sup.2B, --C(O)R.sup.2C,
--C(O)--OR.sup.2C, --C(O)NR.sup.2AR.sup.2B, --OR.sup.2D,
--NR.sup.2AC(O)R.sup.2C, --NR.sup.2AC(O)OR.sup.2C,
--NR.sup.2AOR.sup.2C, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl.
19.-22. (canceled)
23. The compound of claim 1, wherein R.sup.2 is substituted 5 to 6
membered heteroaryl.
24. The compound of claim 1, wherein R.sup.2 is
R.sup.23-substituted 5 to 6 membered heteroaryl; and R.sup.23 is
independently halogen, --CX.sup.23.sub.3, --CHX.sup.23.sub.2,
--CH.sub.2X.sup.23, --OCX.sup.23.sub.3, --OCH.sub.2X.sup.23,
--OCHX.sup.23.sub.2, --CN, --SO.sub.n23R.sup.100D,
--SO.sub.v23NR.sup.100AR.sup.100B, --NHC(O)NR.sup.100AR.sup.100B,
--N(O).sub.m23, --NR.sup.100AR.sup.100B, --C(O)R.sup.100C,
--C(O)--OR.sup.100C, --C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --NR.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.23
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.100A, R.sup.100B, R.sup.100C,
and R.sup.100D are independently hydrogen, --CX.sub.3, --CN,
--COOH, --CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.100A and
R.sup.100B substituents bonded to the same nitrogen atom may
optionally be joined to form a substituted or unsubstituted
heterocycloalkyl or substituted or unsubstituted heteroaryl; X and
X.sup.23 are independently --F, --Cl, --Br, or --I; n23 is
independently an integer from 0 to 4; m23 and v23 are independently
1 or 2; and z23 is an integer from 0 to 3.
25. The compound of claim 1 having the formula: ##STR00082##
wherein ring A is a cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl; R.sup.23 is independently halogen, --CX.sup.23.sub.3,
--CHX.sup.23.sub.2, --CH.sub.2X.sup.23, --OCX.sup.23.sub.3,
--OCH.sub.2X.sup.23, --OCHX.sup.23.sub.2, --CN,
--SO.sub.n23R.sup.100D, --SO.sub.v23NR.sup.100AR.sup.100B,
--NHC(O)NR.sup.100AR.sup.100B, --N(O).sub.m23,
--NR.sup.100AR.sup.100B, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --NR.sup.100A C(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.23
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.100A, R.sup.100B, R.sup.100C,
and R.sup.100D are independently hydrogen, --CX.sub.3, --CN,
--COOH, --CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.100A and
R.sup.100B substituents bonded to the same nitrogen atom may
optionally be joined to form a substituted or unsubstituted
heterocycloalkyl or substituted or unsubstituted heteroaryl; X and
X.sup.23 are independently --F, --Cl, --Br, or --I; n23 is
independently an integer from 0 to 4; m23 and v23 are independently
1 or 2; and z23 is an integer from 0 to 3; wherein -(ring
A)-(R.sup.23).sub.z23 is not ##STR00083##
26. The compound of claim 25, wherein (a) ring A is a heteroaryl,
or (b) ring A is a 5 to 6 membered heteroaryl.
27.-30. (canceled)
31. The compound of claim 23, wherein R.sup.23 is independently
halogen, --CX.sup.23.sub.3, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --NR.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl; two adjacent R.sup.23 substituents may optionally be
joined to form a substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
32. (canceled)
33. The compound of claim 25, wherein R.sup.23 is substituted or
unsubstituted phenyl.
34. (canceled)
35. (canceled)
36. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically acceptable excipient.
37. A method of inhibiting MDA-9 protein activity, said method
comprising contacting the MDA-9 protein with an effective amount of
a PDZ1 domain binder, thereby inhibiting MDA-9 activity, wherein
the PDZ1 domain binder is a compound of claim 1.
38. (canceled)
39. (canceled)
40. The method of claim 37, wherein said compound binds a PDZ1
domain with a Kd of less than 23 .mu.M.
41. (canceled)
42. (canceled)
43. A method of treating cancer in a subject in need thereof, said
method comprising administering to said subject an effective amount
of a PDZ1 domain binder, wherein the PDZ1 domain binder is a
compound of claim 1.
44. (canceled)
45. (canceled)
46. The method of claim 43, wherein said compound binds a PDZ1
domain with a Kd of less than 25 .mu.M.
47. (canceled)
48. (canceled)
49. The method of claim 43, further comprising administering to
said subject an anti-cancer agent.
50. The method of claim 43, wherein said cancer is associated with
increased MDA-9 gene expression.
51. The method of claim 43, wherein said cancer is melanoma,
glioblastoma, head and neck cancer, urothelial cancer, breast
cancer, uveal melanoma, gastric cancer, lung adenocarcinoma,
hepatocellular carcinoma, colorectal cancer, prostate cancer,
pancreatic cancer, or neuroblastoma.
52. The method of claim 43, wherein treating the cancer comprises
inhibiting metastasis of cancer cells in the subject.
53.-59. (canceled)
60. The method of claim 43, wherein treating the cancer comprises
inhibiting cancer associated angiogenesis in the subject.
61.-67. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims to the benefit of U.S. Provisional
Application No. 62/421,468, filed Nov. 14, 2016, and U.S.
Provisional Application No. 62/424,571, filed Nov. 21, 2016, which
are incorporated herein in their entirety and for all purposes.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] The Sequence Listing written in file 052893-502001WO
Sequence Listing_ST25.TXT, created Nov. 13, 2017, 1,360 bytes,
machine format IBM-PC, MS-Windows operating system, is hereby
incorporated by reference.
BACKGROUND
[0004] Described herein, inter alia, are small molecule inhibitors
for the treatment of cancer, including small molecule inhibitors
capable of treating or preventing cancer invasion, attachment
and/or metastasis, for example difficult to treat tumors such as
glioblastoma.
[0005] Cancer (malignant neoplasia) is a disease involving
unregulated cell growth. In cancer, cells divide and grow
uncontrollably, forming malignant tumors, which may invade nearby
parts of the body. The cancer may also spread to more distant parts
of the body through the lymphatic system or bloodstream
(metastasis). Metastasis is a complex series of steps in which
cancer cells leave the primary tumor site, migrate and colonize to
distant sites or organs of the body via the bloodstream or the
lymphatic system. Cancer is usually treated with one or a
combination of chemotherapy, radiation therapy and/or surgery.
While treatment methods have advanced significantly, the outcomes
for particular cancers are still not optimal, current treatments
often have very harsh side effects, and if the cancer is not
detected early, the chances of survival are greatly reduced.
Invasive, metastatic cancer is particularly difficult to treat.
[0006] Glioblastoma multiforme (GBM) is an especially intractable
tumor despite therapeutic advances principally because of its
invasive properties. Radiation is a staple in modern therapeutic
regimens. However, when glioblastoma multiforme (GBM) cells are
irradiated (a common mode of therapy after tumor debulking) the
invasive ability of GBM is increased, and cells surviving radiation
become even more aggressive and invasive.
[0007] There is a need in the art for additional agents to treat
cancer. In particular, there is a need for additional agents that
prevent cancer invasion, attachment and/or metastasis, and that
prevent or attenuate the increase in invasive ability of cancer
cells after exposure to irradiation. Described herein are solutions
to these and other problems in the art.
BRIEF SUMMARY
[0008] In an aspect is provided a compound, or pharmaceutically
acceptable salt thereof, having the formula:
##STR00001##
[0009] Ring B is a cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl.
[0010] R.sup.1 is independently halogen, --CX.sup.1.sub.3,
--CHX.sup.1.sub.2, --CH.sub.2X.sup.1, --OCX.sup.1.sub.3,
--OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN, --SO.sub.n1R.sup.1D,
--SO.sub.v1NR.sup.1AR.sup.1B, --NHC(O)NR.sup.1AR.sup.1B,
--N(O).sub.m1, --NR.sup.1AR.sup.1B, --C(O)R.sup.1C,
--C(O)--OR.sup.1C, --C(O)NR.sup.1AR.sup.1B, --OR.sup.1D,
--NR.sup.1ASO.sub.2R.sup.1D, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)OR.sup.1C, --NR.sup.1AOR.sup.1C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.1
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl;
[0011] L.sup.1 is a bond, --S(O).sub.2--, --N(R.sup.3)--, --O--,
--S--, --C(O)--, --C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--,
--N(R.sup.3)C(O)NH--, --NHC(O)N(R.sup.3)--,
--S(O).sub.2N(R.sup.3)--, --N(R.sup.3)S(O).sub.2--,
--(O)S(O).sub.2N(R.sup.3)--, --N(R.sup.3)S(O).sub.2C(O)--,
--C(O)O--, --OC(O)--, substituted or unsubstituted alkylene,
substituted or unsubstituted heteroalkylene, substituted or
unsubstituted cycloalkylene, substituted or unsubstituted
heterocycloalkylene, substituted or unsubstituted arylene, or
substituted or unsubstituted heteroarylene;
[0012] R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --C(O)R.sup.3C,
--C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0013] R.sup.5 is independently hydrogen,
halogen, --CX.sup.5.sub.3, --CHX.sup.5.sub.2, --CH.sub.2X.sup.5,
--OCX.sup.5.sub.3, --OCH.sub.2X.sup.5, --OCHX.sup.5.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0014] R.sup.6 is independently hydrogen,
halogen, --CX.sup.6.sub.3, --CHX.sup.6.sub.2, --CH.sub.2X.sup.6,
--OCX.sup.6.sub.3, --OCH.sub.2X.sup.6, --OCHX.sup.6.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0015] R.sup.5 and R.sup.6 substituents may optionally be joined to
form a substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0016] R.sup.1A, R.sup.1B, R.sup.1C, R.sup.1D, R.sup.3A, R.sup.3B,
and R.sup.3C are independently hydrogen, --CX.sub.3, --CN, --COOH,
--CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.1A and R.sup.1B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl; R.sup.3A and R.sup.3B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl;
[0017] X, X.sup.1, X.sup.3, X.sup.5, and X.sup.6 are independently
--F, --Cl, --Br, or --I; n1 is independently an integer from 0 to
4; m1 and v1 are independently 1 or 2; and z1 is an integer from 0
to 5.
[0018] In an aspect is provided a compound, or pharmaceutically
acceptable salt thereof, having the formula:
##STR00002##
[0019] R.sup.1 is independently halogen, --CX.sup.1.sub.3,
--CHX.sup.1.sub.2, --CH.sub.2X.sup.1, --OCX.sup.1.sub.3,
--OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN, --SO.sub.n1R.sup.1D,
--SO.sub.v1NR.sup.1AR.sup.1B, --NHC(O)NR.sup.1AR.sup.1B,
--N(O).sup.m1, --NR.sup.1AR.sup.1B, --C(O)R.sup.1C,
--C(O)--OR.sup.1C, --C(O)NR.sup.1AR.sup.1B, --OR.sup.1D,
--NR.sup.1ASO.sub.2R.sup.1D, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)OR.sup.1C, --NR.sup.1AOR.sup.1C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.1
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl;
[0020] R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--SO.sub.n2R.sup.2D, --SO.sub.v2NR.sup.2AR.sup.2B,
--NHC(O)NR.sup.2AR.sup.2B, --N(O).sub.m2, --NR.sup.2AR.sup.2B,
--C(O)R.sup.2C, --C(O)--OR.sup.2C, --C(O)NR.sup.2AR.sup.2B,
--OR.sup.2D, --NR.sup.2ASO.sub.2R.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O)OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0021] L.sup.1 is a bond, --S(O).sub.2--, --N(R.sup.3)--, --O--,
--S--, --C(O)--, --C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--,
--N(R.sup.3)C(O)NH--, --NHC(O)N(R.sup.3)--,
--S(O).sub.2N(R.sup.3)--, --N(R.sup.3)S(O).sub.2--,
--(O)S(O).sub.2N(R.sup.3)--, --N(R.sup.3)S(O).sub.2C(O)--,
--C(O)O--, --OC(O)--, substituted or unsubstituted alkylene,
substituted or unsubstituted heteroalkylene, substituted or
unsubstituted cycloalkylene, substituted or unsubstituted
heterocycloalkylene, substituted or unsubstituted arylene, or
substituted or unsubstituted heteroarylene;
[0022] R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --C(O)R.sup.3C,
--C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0023] L.sup.2 is a bond, --S(O).sub.2--, --N(R.sup.4)--, --O--,
--S--, --C(O)--, --C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--,
--N(R.sup.4)C(O)NH--, --NHC(O)N(R.sup.4)--,
--S(O).sub.2N(R.sup.4)--, --N(R.sup.4)S(O).sub.2--,
--(O)S(O).sub.2N(R.sup.4)--, --N(R.sup.4)S(O).sub.2C(O)--,
--C(O)O--, --OC(O)--, substituted or unsubstituted alkylene,
substituted or unsubstituted heteroalkylene, substituted or
unsubstituted cycloalkylene, substituted or unsubstituted
heterocycloalkylene, substituted or unsubstituted arylene, or
substituted or unsubstituted heteroarylene;
[0024] R.sup.4 is independently hydrogen, --CX.sup.4.sub.3,
--CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN, --C(O)R.sup.4C,
--C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl;
[0025] R.sup.1A, R.sup.1B, R.sup.1C, R.sup.1D, R.sup.2A, R.sup.2B,
R.sup.2C, R.sup.2D, R.sup.3A, R.sup.3B, R.sup.3C, R.sup.4A,
R.sup.4B, and R.sup.4C are independently hydrogen, --CX.sub.3,
--CN, --COOH, --CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.1A and R.sup.1B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl; R.sup.2A and R.sup.2B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl; R.sup.3A and R.sup.3B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl; R.sup.4A and R.sup.4B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl; X, X.sup.1, X.sup.2,
X.sup.3, and X.sup.4 are independently --F, --Cl, --Br, or --I; n1
and n2 are independently an integer from 0 to 4; m1, m2, v1, and v2
are independently 1 or 2; and z1 is an integer from 0 to 4.
[0026] In another aspect is provided a method of preventing or
treating cancer in a subject in need thereof, including
administering to the subject a therapeutically effective amount of
a compound described herein, including embodiments thereof, wherein
the therapeutically effective amount is sufficient to prevent or
treat the cancer.
[0027] In another aspect is provided a method of sensitizing cancer
cells to killing by radiation, including contacting the cancer
cells with an effective (e.g., therapeutically effective) amount of
a compound described herein, including embodiments thereof, wherein
the therapeutically effective amount is sufficient to sensitize the
cancer cells to killing by radiation.
[0028] In another aspect is provided a method of slowing or
preventing metastasis of cancer cells in a subject in need thereof,
including administering to the subject an effective (e.g.,
therapeutically effective) amount of a compound described herein,
including embodiments thereof, wherein the therapeutically
effective amount is sufficient to slow or prevent the
metastasis.
[0029] In another aspect is provided s a method of treating a
glioblastoma multiforme brain tumor in a subject in need thereof,
including performing surgery on the subject to debulk the
glioblastoma multiforme brain tumor; radiosensitizing remaining
tumor cells by administering to the subject a therapeutically
effective amount of at least one of the compounds of any of the
invention, wherein the therapeutically effective amount is
sufficient to sensitize the remaining tumor cells to killing by
radiation; and providing radiation therapy to the subject.
[0030] In an aspect is provided a pharmaceutical composition
including a compound as described herein, including embodiments
thereof, and a pharmaceutically acceptable excipient.
[0031] In an aspect is provided a method of inhibiting MDA-9
protein activity, the method including contacting the MDA-9 protein
with an effective amount of a PDZ1 domain binder, thereby
inhibiting MDA-9 activity.
[0032] In an aspect is provided a method of treating cancer in a
subject in need thereof, the method including administering to the
subject an effective amount of a PDZ1 domain binder.
[0033] In an aspect is provided a method of preventing metastasis
of cancer cells in a subject in need thereof, the method including
administering to the subject an effective amount of a PDZ1 domain
binder.
[0034] In an aspect is provided a method of inhibiting cancer
associated angiogenesis in a subject in need thereof, the method
including administering to the subject an effective amount of a
PDZ1 domain binder.
[0035] In an aspect is provided a method of treating an
inflammatory disease in a subject in need thereof, the method
including administering to the subject an effective amount of a
PDZ1 domain binder.
[0036] In an aspect is provided a method of treating a
neurodegenerative disease in a subject in need thereof, the method
including administering to the subject an effective amount of a
PDZ1 domain binder.
[0037] In an aspect is provided a method of treating an infectious
disease in a subject in need thereof, the method including
administering to the subject an effective amount of a PDZ1 domain
binder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A-1C. MDA-9/Syntenin is involved in radiosensitivity.
FIG. 1A. The REMBRANDT database was mined for glioma patients with
no previous radiation treatment, who then underwent radiation
therapy. These were stratified by SDCBP (mda-9/syntenin) expression
(High was set >1.5-fold overexpression). High tumor
MDA-9/Syntenin led to a worse prognosis in those undergoing
radiotherapy. FIG. 1B. U1242-shcon and U1242-shmda-9 cells were
analyzed for colony formation 14 d post radiation treatment. FIG.
1C. U1242 cells treated with either Ad.5/3-shcon or Ad.5/3-shmda-9
were analyzed for viability at the indicated time points via MTT
assay. Error bars=.+-.s.d. *p<0.05, **p<0.01.
[0039] FIG. 2. MDA-9/Syntenin knockdown inhibits radiation-induced
invasion. U87, U251, U1242, and GBM6 cells were treated with either
Ad.5/3-shcon or Ad.5/3-shmda-9 and irradiated 48 hrs later. These
cells were then seeded in a trans-well Matrigel invasion assay and
stained after 18 hr. Relative invasion is quantified from 5 random
fields. Error bars=.+-.s.d. *p<0.05, **p<0.01.
[0040] FIGS. 3A-3F. Inhibition of MDA-9/Syntenin impairs Src-EphA2
signaling. FIGS. 3A-3C. Immunoblot analysis of GBM cells treated
with either Ad.5/3-shcon or Ad.5/3-shmda-9 and irradiated 48 hrs
later. Cell lysates were collected 24 hr post-radiation. Changes in
protein levels were quantified in FIGS. 3D-3F. .beta.-actin used as
protein loading control.
[0041] FIGS. 4A-4E. FIG. 4A. Chemical structures of the two initial
fragment hits (top) and the structure of the resulting molecule
113B7 (PDZ1i). FIG. 4B. Superposition of 2D [.sup.15N,
.sup.1H]-HSQC spectra of 50 .mu.M MDA-9 PDZ12 tandem domain in the
absence and presence of 350 .mu.M113B7. FIG. 4C. Binding curve and
representative spectra for the titration of 113B7 against MDA-9
PDZ12 tandem domain. Top: Zoom of box region in FIG. 4B after
titrating 113B7 into Mda9 PDZ tandem domain. The cross peak
corresponding to the backbone amide of residue A147 experiences a
gradual shift upon the addition of 113B7. Bottom: Titration curve
of 113B7 for residue A147. The dissociation constant for the
binding of 113B7 to MDA-9 was calculated to be 21.37.+-.8.62 .mu.M.
FIG. 4D. Superposition of 2D [.sup.15N, .sup.1H]-HSQC spectra of 50
.mu.M PDZ domain from X11/mint scaffold protein (20 mM; 33%
identity with PDZ1) are shown in absence and in presence of 100 mM
113B7. No appreciable binding is detected. FIG. 4E. Docked
structure of 113B7 in complex with MDA-9 PDZ tandem domain (PDB:
1W9E). The pose was obtained with GOLD. Two PDZ domains of MDA-9
are labeled. The surface of MDA-9 PDZ tandem domain is displayed.
The structure of 113B7 is displayed in balls and sticks.
[0042] FIG. 5. Docked structure of PDZ1i (113B7) in complex with
MDA-9 PDZ tandem domain (PDB: 1W9E) and chemical structure of PDZ1i
(113B7). The pose was obtained with GOLD. Two PDZ domains of MDA-9
are labeled. The surface of MDA-9 PDZ tandem domain is displayed.
The structure of 113B7 is displayed in balls and sticks.
[0043] FIG. 6. Synthetic scheme for the preparation of 113B7
(PDZ1i). Reagents and Conditions: (a) HATU, DIEA, DMSO, r.t., 24 h;
(b) HATU, DIEA, DMF, r.t., 24 h.
[0044] FIGS. 7A-7B. FIG. 7A. Im-PHFA, primary immortal human fetal
astrocyte cells were infected with an MDA-9/Syntenin expression
plasmid carrying adenovirus. 48 hrs post-infection both the control
and MDA-9/Syntenin overexpressing cells were treated with DMSO or
PDZ1i followed by seeding in a trans-well Matrigel invasion assay
and stained after 24 hrs. FIG. 7B. T98G and U87 were treated with
DMSO or PDZ1i and seeding in a trans-well Matrigel invasion assay
and stained after 24 hrs. Invasion was quantified from 4 random
fields. Data represents fold changes and average.+-.S.D.
[0045] FIGS. 8A-8D. PDZ1i produces similar effects to
mda-9/syntenin knockdown post-radiation. FIG. 8A. ImPHFA and U87
cells were treated with 50 .mu.M PDZ1i 2 h prior to radiation and
analyzed for colony formation after 14 d. FIG. 8B. U87 cells were
treated with 50 .mu.M PDZ1i 2 h prior to radiation and analyzed via
MTT assay after 24 h. FIG. 8C. U87 cells were pretreated with 50
.mu.M PDZ1i 2 h prior to radiation and subsequently seeded in a
trans-well Matrigel invasion assay and stained after 24 h. Invasion
was quantified from 5 random fields in FIG. 8D. Error bars=.+-.s.d.
*p<0.05, **p<0.01.
[0046] FIGS. 9A-9C. PDZ1i treatment impairs EGFRvIII and FAK
signaling, as well as EGFRvIII-MDA-9 interaction. FIG. 9A.
Immunoblot analysis showing expression of mutated EGFR in U87 or
U87EGFRvIII cells. FIG. 9B. Cells were treated with either DMSO or
PDZ1i 2 hrs prior to radiation. These cells were subsequently
seeded on fibronectin-coated plates and cell lysates were collected
after 1 hr and analyzed for protein expression. FIG. 9C. Lysates
from cells treated with and without radiation or PDZ1i as indicated
were analyzed via immunoprecipitation. Anti-MDA-9, top, and
anti-EGFR, bottom, were used to pull down associated complexes, and
western blotting was performed with the indicated antibodies. Left
lanes include IgG and control input samples.
[0047] FIGS. 10A-10L. PDZ1i inhibits key invasion proteins. U1242
cells were treated with either DMSO or 50 .mu.M PDZ1i 2 hrs prior
to radiation in serum-free media. After 48 hrs, media was collected
analyzed via the Proteome Profiler Human Protease Array Kit
(R&D Systems). Relative protein amounts of the indicated
proteins were quantified via ImageJ. FIG. 10A, MMP1; FIG. 10B,
MMP2; FIG. 10C, MMP3; FIG. 10D, MMP8; FIG. 10E, MMP9; FIG. 10F,
MMP13; FIG. 10G. Cathepsin A; FIG. 10H, Cathepsin B; FIG. 10I,
Cathepsin C; FIG. 10J, Cathepsin S; FIG. 10K, Cathepsin V; FIG.
10L, ASAM9.
[0048] FIG. 11. PK studies with PDZ1i. 3.0 mg/Kg (I.V.) and 30.0
mg/Kg (I.P.) drug were administered in mice (n=3) and compound
concentration in serum were measured at the indicated times.
Noteworthy are the lack of adverse signs of toxicity during the
experiment, the very slow clearance with T1/2>9 hr in each route
and the 80% bioavailability for the compounds administered LP.
[0049] FIG. 12. PDZ1i crosses the blood brain barrier (BBB).
Primary human malignant glioma cells (GBM6) were seeded on the
bottom well of a transwell chamber. In the top chamber, a monolayer
of HBMEC cells separated GBM6 cells from media containing DMSO, 30
.mu.M PDZ1i or Temozolomide (500 .mu.M). After 24 h incubation in
these conditions, invasion of GBM6 cells were analyzed by seeding
in a trans-well Matrigel invasion assay and stained after 24 h. NB:
PDZ1i treatment and invasion assay without HBMEC barrier. Error
bars=.+-.s.d.
[0050] FIGS. 13A-13E. Effect of PDZ1i on survival in an in vivo
model of glioma. GBM6 cells were pretreated for 2 hrs prior to
intracranial injection with vehicle (DMSO) or PDZ1i and after 7
days, brain tissue was isolated and sectioned at injection site.
FIG. 13A, brain tissue at site of injection of DMSO treated cells;
FIG. 13B, brain tissue are site of injection of PDZ1i treated
cells. 7 days after tumor implantation, mice received either
vehicle or PDZ1i (30 mg/kg) three times per week for 3 weeks. Brain
tissue was isolated and analyzed via H&E stain. FIG. 13C, brain
tissue from mice treated with vehicle; FIG. 13D, brain tissue from
mice treated with PDZ1i. FIG. 13E, Kaplan-Myer curves of these
groups based on animal survival. **p<0.01.
[0051] FIGS. 14A-14F. PDZ1i treatment combined with radiation in an
in vivo model of GBM. FIG. 14A. U1242-luc cells were injected
intracranially into nude mice. After 7 days mice were randomized to
4 groups, and mice receiving therapy were treated on days 11-14 as
pictured. FIG. 14B. Kaplan-Meier survival curves for each treatment
group. Median survival as listed. Two brain tissue samples were
isolated for each group, sectioned, and H and E staining is shown.
FIG. 14C, DMSO treated mice, FIG. 14D, PDZ1i treated mice, FIG.
14E, mice treated with radiation and DMSO; FIG. 14F, mice treated
with radiation and PDZ1i.
[0052] FIGS. 15A-15D. MDA-9 regulates PC progression. FIG. 15A)
mda-9 expression level in primary immortal normal prostate
epithelial (RWPE-1) and different PC cells. FIG. 15B) Knockdown of
mda-9 by shRNA reduces in vitro invasive properties of different PC
cells. FIG. 15C) over expression of mda-9/syntenin in RWPE-1 cells
enhances invasiveness. FIG. 15D) Lucifearase expressing ARCaPM
cells either carrying control shRNA or shmda-9 were inoculated i.v.
into a thymic nude mice by tail vein injections. Mice were
maintained until tumors reached maximally permitted size, then
animals were euthanized. Kalpan-Meier survival graph was
constructed using Graph Pad software.
[0053] FIG. 16A-16N. IGFBP-2 regulates STAT3 activity in PC cells.
FIG. 16A) Western blot analysis was performed for the indicated
cells for both phospho-STAT3 (Tyr705) and total STAT3. FIG. 16B)
Cells were either transfected with control vector, mda-9 expression
vector (RWPE-1) or mda-9 shRNA vector (PC-3 and DU145). 48 h after
transfection, cells were replated on fibronectin-coated plates for
1 h. Western blot analysis was conducted with the indicated
antibodies. In FIGS. 16C-16E) The indicated cells, FIG. 16C),
RWPE-1, FIG. 16D), PC-3 and FIG. 16E), DU145, were co-transfected
with a reporter gene and empty vector, mda-9 or shmda-9 as
indicated in the figure and after 48 h, luciferase activity was
measured. Data presented as fold-change in comparison with the
control group (empty vector). FIG. 16F) Cells were co-transfected
with different expression plasmids as indicated. 48 h later, cells
were trypsinized and invasion was assayed. Cells were counted using
bright field microscopy. FIG. 16G) Cells were transfected with
reporter genes and after 36 h, cells were treated with Brefeldin A
for 30 min. Media was removed and cells were cultured for an
additional 3 h in serum-free media and luciferase activity was
measured. FIG. 16H) expression of IGFBP-2 mRNA in different PC
cells as determined by qPCR. FIGS. 16I-16J) Cells (FIG. 16I, P-69
and RWPE-1, FIG. 16J, PC-3 and DU-145) were co-transfected with
reporter genes and different plasmids and after 48 h luciferase
activity was measured. Data presented as fold-change after
normalizing with renilla luciferase activity. FIG. 16K) RWPE-1
cells were treated with rIGFBP-2 under different conditions as
indicated in the figure and phospho-IGF-1R expression was
determined by Western blotting. FIG. 16L) 200 fag of total protein
from PC-3 cells was incubated with MDA-9 overnight for
immunoprecipitation and Western blotting was performed with the
anti-IGF-1R antibody to confirm interaction. FIG. 16M) RWPE-1 cells
were transfected with wild type or mutant mda-9 vectors. 48 h
later, cells were replated on fibronectin-coated plates for 1 h and
cell lysates were analyzed using the indicated antibodies. FIG.
16N) Model of MDA-9/syntenin-mediated PC progression.
[0054] FIGS. 17A-17H. Effect of PDZ1i on invasion, MDA-9/IGF-R1
interaction and IGF-R1, STAT3 and SRC phosphorylation. FIG. 17A)
Different cancer cells (25,000 cells/well) from various anatomic
origins were pre-treated with either DMSO (vehicle) or PDZ1i (dose
as indicated) and invasion ability was assayed using modified
Boyden Chamber according to the manufacturer's instructions.
Photomicrographs were taken at 10.times. magnification and
quantification of the results of three independent experiments is
provided in the graphs. The data presents mean.+-.S.D. B) The
designated cells were treated with DMSO or PDZ1i and invasion
properties were determined using a modified Boyden Chamber assay
(BD bioscience). RWPE-1 mda-9: mda-9 transiently overexpressing
cell line. FIG. 17C) and FIG. 17D) cell lysates prepared from DU145
cells, treated or not with PDZ1i, were subjected to OP using
anti-MDA-9 antibody and IB was performed using anti-IGF-1R
antibody. FIG. 17E) Cells were growth starved for 24 h and treated
with either DMSO or PDZ1i (dose indicated in .mu.M) for 6 h. Cells
were treated with human recombinant IGFBP-2 (hIGFBP-2, 10 ng/ml)
for 2 h, lysates were prepared and western blot analysis was
conducted with specific antibodies. FIG. 17F) Cells were
serum-starved for 24 h and treated with either DMSO or PDZ1i (20
.mu.M) for 6 h. Cells were treated with human recombinant IGFBP-2
(hIGFBP-2, 10 ng/ml) for different times (30 to 120 min), cell
lysates were prepared and subjected to western blotting. FIG. 17G)
Cells were serum-starved for 24 h and treated with either DMSO or
PDZ1i (20 .mu.M) for 6 h. Cells were treated with human recombinant
IL-6 (hIGFBP-2, 1 ng/ml) for 2 h, cell lysates were prepared and
analyzed by western blotting. FIG. 17H) the indicated cells were
treated with DMSO or PDZ1i (50 .mu.M) for 24 h. Tumor-derived
conditioned media were subjected to western blotting analysis (left
panel) for the expression of MMP-2 and MMP-9 and Zymography (right
panel) for enzymatic activity.
[0055] FIGS. 18A-18C. PDZ1i suppresses production of tumor-derived
pro-angiogenic factors. FIG. 18A) The strategy for qPCR based array
is shown schematically. Briefly, ARCaPM cells were pre-treated with
either DMSO or PDZ1i (50 .mu.M) for 24 h followed by RNA
extraction. cDNA was prepared and angiogenesis-related gene
expression arrays were analyzed. FIG. 18B) Cell lines were treated
with DMSO or PDZ1i for 24 h and qPCR was performed for the specific
indicated genes using Taqman probes. FIG. 18C) Cells were treated
with DMSO or PDZ1i for 24 h and expression of VEGF-A mRNA was
analyzed.
[0056] FIG. 19A-19F. PDZ1i suppresses prostate cancer metastasis
and tumor progression. FIG. 19A) Survival data for athymic nude
mice in which DMSO or PDZ1i pre-treated ARCaPM cells were injected.
FIG. 19B) Survival data for athymic nude mice which were injected
with (n=5, each group) ARCaPM-Luc (1.times.10.sup.6 cells in 100
.mu.l saline) through an intracardiac route. Mice received either
vehicle or PDZ1i every alternative day (9 injections during the
first three months) and maintained until they needed to be
euthanized. FIG. 19C) Survival data for C57BL/6 mice injected
through the intracardiac route with RM1-Luc. Vehicle or PDZ1i was
administered through intraperitoneal injection every alternate day
(total 3 injections during the first week). Survival curves were
generated using 6 control vehicle-treated and 5 PDZ1i-treated.
Kaplan-Meier survival curves were prepared using Graph Pad
software. FIG. 19D) PDZ1i can efficiently inhibit tumor progression
in Hi-Myc mice. Graphical representation of the average prostate
weights from control and treated groups. FIG. 19E and FIG. 19F)
Photomicrographs representing the histological changes in prostate
sections obtained from 6-month old Hi-Myc mice receiving either
vehicle (FIG. 19E) or PDZ1i (FIG. 19F).
[0057] FIGS. 20A-20C. MDA-9 expression is elevated in breast
cancer. (FIG. 20A) Immunohistochemistry of breast cancer patient
samples showing overexpression of MDA-9 in breast cancer. (FIG.
20B) MDA-9 protein expression is increased in invasive and
metastatic cell lines. (FIG. 20C) MDA-9 transcript expression is
increased in invasive and metastatic breast cancer cell lines.
[0058] FIGS. 21A-21C. MDA-9 enhances invasion and cytoskeletal
rearrangement. (FIG. 21A) Western blots showing efficient silencing
of MDA-9 in MDA-MB-231 cells, efficient silencing of MDA-9 in
SUM159 cells and efficient over expression of MDA-9 in T47D cells.
(FIG. 21B) Graphical representation of the invasion assay results
in MDA-9 silenced MDA-MB-231 and SUM159 cells and MDA-9
overexpressing T47D cells. (FIG. 21C) Representative images showing
change in cytoskeletal reorganization following silencing of MDA-9
expression in MDA-MB-231 and SUM159 cells and overexpressing MDA-9
in T47D cells. *, p<0.05; ***, p<0.0001.
[0059] FIGS. 22A-22B. Silencing or overexpressing MDA-9 regulates
EMT. (FIG. 22A) Representative images showing change in morphology
in 3D culture following silencing MDA-9 expression in MDA-MB-231
and SUM139 cells and overexpressing MDA-9 in T47D cells. (FIG. 22B)
Western blots showing changes in key epithelial and mesenchymal
markers following modulation of MDA-9 expression.
[0060] FIGS. 23A-23D. MDA-9 modulates small GTPases RhoA and Cdc42
via TGF.beta.1. Fold change in active cdc42 and RhoA levels in
(FIG. 23A) MDA-MB-231 cells and (FIG. 23B) SUM159 cells after
silencing MDA-9 expression and after re-introduction of TGF.beta.1.
(FIG. 23C) Fold change in active cdc42 and RhoA levels in T47D
cells after overexpression of MDA-9 and after addition of
TGF.beta.1 inhibitor (FIG. 23D) Western blots showing changes in
TGF.beta.1 expression following modulation of MDA-9 expression. *,
p<0.05; **, p<0.01; 0.0001.
[0061] FIGS. 24A-24C. TGF.beta.1 modulation in MDA-9 modulated
cells regulates invasion and cytoskeletal rearrangement. Graphical
representation of the invasion assay results and representative
images showing cytoskeletal rearrangement upon re-introduction of
TGF.beta.1 in MDA-9 silenced MDA-MB-231 (FIG. 24A) and SUM159 (FIG.
24B) cells. Inset: western blots showing efficient silencing of
MDA-9. (FIG. 24C) Graphical representation of the invasion assay
results and representative images showing cytoskeletal
rearrangement upon TGF.beta.1 inhibition in T47D cells
overexpressing MDA-9. Inset: western blot showing efficient
overexpression of MDA-9. ***, p<0.0001.
[0062] FIGS. 25A-25D. MDA-9 interacts with TGF.beta.1. (FIG. 25A)
Immunoprecipitation with MDA-9 antibody and immunoblotting using
TGF.beta.1 antibody showed that MDA-9 interacts with TGF.beta.1.
(FIG. 25B) Immunoprecipitation with TGF.beta.1 tag antibody and
immunoblotting with MDA-9 antibody showed that TGF.beta.1 interacts
with MDA-9 and this interaction was decreased when MDA-9 expression
was silenced. (FIG. 25C) Schematic representation of full length
MDA-9 constructs and PDZ1 deleted constructs (with FLAG tag). (FIG.
25D) Immunoprecipitation with FLAG antibody and immunoblotting with
TGF.beta.1 antibody shows that PDZ1 domain of MDA-9 interacts with
TGF.beta.1.
[0063] FIGS. 26A-26G. Silencing MDA-9 causes a reduction in lung
metastasis, which can be rescued by restoration of TGF.beta.1
expression. (FIG. 26A) Western blot images showing stable
expression of TGF.beta.1 in MDA-MB-231 control TGF.beta.1
luciferase cells and MDA-MB-231 shMDA-9 TGF.beta.1 luciferase
cells. (FIG. 26B) Bioluminescence imaging showing reduction of lung
metastasis in mice injected with MDA-MB-231 shMDA-9 luciferase
cells and rescue following re-expression of TGF.beta.1. (FIG. 26C)
Bioluminescence imaging showing rescue of lung metastasis in mice
injected with MDA-MB-231 shMDA-9 TGF.beta.1 luciferase cells
compared to mice injected with MDA-MB-231 shMDA-9 luciferase cells.
(FIG. 26D) Bioluminescent images of the lungs showing metastasis.
(FIG. 26E) Western blot images of cells isolated from the
respective lungs and probed for MDA-9 and TGF.beta.1 tag
expression. (FIG. 26F) H&E images of the lung sections showing
presence of lung metastases. MDA-MB-231 control luciferase and
MDA-MB-231 control TGF.beta.1 luciferase cells efficiently
colonized the entire lungs. MDA-MB-231 shMDA-9 luciferase cells
formed a few small lung metastases while MDA-MB-231 shMDA-9
TGF.beta.1 luciferase cells showed partial restoration of
metastatic capabilities and formed multiple larger lung metastatic
lesions. (FIG. 26G) Schematic representation of the signaling
mechanism mediated by MDA-9 to regulate cytoskeletal rearrangement,
EMT and invasion. MDA-9 has previously been shown to regulate the
formation of various integrin .beta.1 signaling complexes. Integrin
.beta.1, in turn, functions to enhance TGF.beta.1-mediated
non-canonical signaling and EMT and blocking integrin .beta.1
function inhibited TGF.beta.-mediated non-canonical signaling and
EMT. In breast cancer cells, MDA-9 interacts with TGF.beta.1 and
regulates the small GTPases RhoA and cdc42 via TGF.beta.1. Further,
MDA-9 regulates EMT and invasion via TGF.beta.1.
[0064] FIG. 27. DNA copy number of MDA-9 is elevated in human
breast cancer patients. Histogram from TCGA database in Oncomine
demonstrating MDA-9 copy number elevation in breast tumors compared
to normal breast.
[0065] FIGS. 28A-28C. Modulation of MDA-9 expression causes changes
in cell shape. Representative images showing change in morphology
in 2-dimensional culture on plastic plates following silencing
MDA-9 expression in (FIG. 28A) MDA-MB-231 and (FIG. 28B) SUM159 and
overexpressing MDA-9 in (FIG. 28C) T47D cells.
[0066] FIG. 29. MDA-9 and TGF.beta.1 are co-expressed in breast
cancer patient samples. Correlation data from TCGA database in
Oncomine demonstrating that MDA-9, TGF.beta.1 and SNAI2 (also known
as Slug, a key EMT marker) genes are coexpressed in breast cancer
patient samples.
[0067] FIG. 30. DNA copy number of TGF.beta.1 is elevated in in
human breast cancer patients. Histogram from TCGA database in
Oncomine demonstrating TGF.beta.1 copy number elevation in breast
tumors compared to normal breast in the same sample set assessed
for MDA-9 expression in FIG. 27.
[0068] FIGS. 31A-31B. Integrin .beta.1 regulates cytoskeletal
reorganization. (FIG. 31A) Representative images showing change in
cytoskeletal reorganization following addition of Integrin .beta.1
blocking antibody in SUM1S9 cells. (FIG. 31B) Representative images
showing change in cytoskeletal reorganization following
overexpression of MDA-9 that was restored upon addition of Integrin
.beta.1 blocking antibody.
[0069] FIG. 32. Cartoon illustration of tumor cells in
microenvironment navigating away from tumor microenvironment.
[0070] FIG. 33. Detailed cartoon illustration of the tumor
microenvironment.
[0071] FIG. 34. Flow chart showing MDA-9 and the metastatic
cascade.
[0072] FIGS. 35A-35C. The expression of mda-9/Syntenin (SDCBP)
(relative to normal samples) in melanoma (FIG. 35A), prostate
cancer (FIG. 35B), and liver cancer (FIG. 35C). The expression
values were derived from public genome-wide expression datasets.
The relative expression (z) is equal to (In-- Average
Inorm)/standard dev norm, where n refers to every sample in the
dataset (including tumors), while norm refers to normal samples
only.
[0073] FIG. 36. Graph showing survival fraction as a function of
days for patients with MDA-9 expression in the lower half and upper
half.
[0074] FIG. 37. MDA-9 promotes tumor angiogenesis. Knockdown of
MDA-9 with shRNAs reduced angiogenesis while over expression of
MDA-9 increased angiogenesis.
[0075] FIG. 38. Crystal structure of MDA-9/Syntenin showing both
PDZ domains and the intermediate (e.g., interface) region between
the domains.
[0076] FIG. 39. Structure of 113B7, also referred to as PDZ1i and
PDZ1in.
[0077] FIG. 40. Cartoon illustration of block of MDA-9 interactions
with downstream proteins upon treatment with PDZ1i.
[0078] FIG. 41. Results of combination treatment (PDZ1i and
anti-cancer agent) in different cancers (GBM, pancreatic cancer,
and HCC).
[0079] FIG. 42. Cancer progression to metastasis: a temporal
process mediated by multiple initiating, progressing and
virulence-mediating genes. This model highlights the properties
elicited by tumorigenic genes vs. the three classes of metastasis
genes (initiation, progression and virulence).
[0080] FIG. 43. Model of mda-9/syntenin mediated induction of
NF-.kappa.B and its downstream genes and processes through its
interaction with c-Src. MDA-9/syntenin interactions with c-Src
assemble c-Src/FAK signaling complexes and leads to activation of
the p38 MAPK/NF-.kappa.B pathway that regulates expression of genes
involved in cell motility and invasion.
[0081] FIG. 44. Hypothetical model of MDA-9/Syntenin-mediated
angiogenesis. MDA-9/Syntenin upon interaction with c-Src, activates
HIF-1.alpha. in an AKT-dependent pathway and induces IGFBP-2
expression. IGFBP2 acts as a chemoattractant for endothelial cells
and induces VEGF-A secretion resulting in angiogenic
phenotypes.
[0082] FIG. 45. Docked structure of compound 113B7 (PDZ1in) on the
surface of MDA-9/Syntenin. The docked structure is supported by NMR
chemical shift mapping data. The titration allows for the
calculation on an upper limit for the dissociation constant of the
complex, Kd<10 .mu.M. 113B7 does not bind appreciably to PDZ2
from MDA-9/Syntenin or other PDZs used as counter screens.
[0083] FIG. 46. Small molecule MDA-9/Syntenin PDZ1in (113B7)
inhibits invasion in melanoma, HCC and prostate carcinoma cells.
Tumor cells were pre-treated with either DMSO (vehicle) or PDZ1in
(113B7) (dose indicated) and invasion ability was assayed using a
modified Boyden Chamber. Results of three independent experiments
is provided in the graphs+S.D. Melanoma: C8161.9, MeWo; PC: PC-3,
ARCaP; HCC: Huh7, QGY7703.
[0084] FIG. 47. mda-9/syntenin induces an invasive phenotype in
normal immortal melanocytes (FM516), prostate epithelial (RWPE-1)
and pancreatic mesenchymal (LT-2) cells, which is inhibited by
113B7. Invasion assay as performed in FIG. 46.
[0085] FIGS. 48A-48C. Biological effects of PDZ1i in vivo in
melanoma, HCC and PC. FIG. 48A) Compounds were administered I.P. 6
times within first two weeks following inoculation (I.V. injection)
of B16 cells in C57BL/6 mice to evaluate the anti-metastatic
efficacy. FIG. 48B) HCC-driven tumor xenograft was established in
athymic nude mice. Compound alone or in combination with Sorafenib
were given through I.P. Tumor volumes were considered as an end
point of this study. FIG. 48C) Hi-Myc, a mouse model for
spontaneous prostate cancer either received the compound or vehicle
at the age of 8 weeks (immediate after onset of disease) for total
9 injections (3/week). After 16 weeks from the first injection,
prostate were collected and pathologically evaluated.
Representative photomicrographs (Left panel) and H and E stained
slides (Right panel) from prostate were presented.
[0086] FIGS. 49A-49D. Effects of PDZ1in on interactions between
MDA-9/Syntenin and its various interacting partners. FIG. 49A)
C8161.9 cells were pre-treated with either DMSO or PDZ1in and cell
lysates were immunoprecipitated and immunoblotted with the
indicated antibodies. FIG. 49B) 200 .mu.g total protein from PC-3
(Prostate Cancer) cells were immunoprecipitated and immunoblotted
with the antibody as indicated. FIG. 49C) Left Panel,
Coimmunoprecipitation studies were done in different conditions to
document AEG-1 and MDA-9/Syntenin interaction in the membrane.
Right panel, co-IP studies to document AEG-1 and EGFR interaction
in the membrane fraction. FIG. 49D), immunofluorescence studies in
non-permeabilized cells to document co-localization of AEG-1,
MDA-9/Syntenin and EGFR in the membrane.
[0087] FIG. 50. Major MDA-9/Syntenin-mediated signaling pathways
that contribute to tumor progression/invasion/metastasis in
multiple cancers. In Melanoma (Left Panel), upon ECM engagement,
FAK and Src complex is recruited in the plasma membrane to initiate
the initial signaling. MDA-9/Syntenin physically interacts with Src
resulting in the formation of multimeric complexes that activate
the NF-.kappa.B pathway and consequently induce downstream proteins
essential for metastasis. In hepatocellular carcinoma (RightPanel),
MDA-9/Syntenin interacts in the plasma membrane with AEG-1 and EGFR
to form a functional unit, which possibly activates NF.kappa.B
pathway through the phosphorylation of Src to stimulate/initiate
tumor progression signaling cascades.
[0088] FIG. 51. Hypothetical model for MDA-9/Syntenin mediated
prostate cancer progression. Initial supportive evidences were
documented that MDA-9/Syntenin and IGF-1R physically interacts and
stabilizes the functional unit to activate the STAT3 through
phopshorylation at the tyrosine 705 position. Phospho-STAT3 forming
a dimer and translocate to nucleus to induce various genes that
actively participate in prostate cancer progression.
[0089] FIG. 52. Left panel, Docked structure of compound 113B7 on
the surface of MDA-9/Syntenin. The docked structure is supported by
NMR chemical shift mapping data. The titration (see later in FIG.
55 for chemical shift mapping and titration) allows for the
calculation of an upper limit for the dissociation constant of the
complex, Kd<10 .mu.M. Of note is that 113B7 does not bind
appreciably to PDZ2 from MDA-9/Syntenin or other PDZs used as
counter screens as can be seen in the right panel spectra. The
[.sup.15N, .sup.1FI]-HSQC spectra of MDA-9/Syntenin PDZ2 only
domain are reported in the top right panel (apo at 20 .mu.M; in
presence of 100 .mu.M113B7). In the bottom right panel can be seen
the spectra of the PDZ domain from X11/mint scaffold protein (33%
identity with PDZ1) are reported in absence (20 .mu.M protein) and
in presence of 100 .mu.M113B7. No appreciable binding is detected
in both cases.
[0090] FIG. 53. PK studies with 113B7. 3.0 mg/Kg (IV) and 30.0
mg/Kg (IP) drag were administered in mice (n=3) and compound
concentration in serum were measured at the indicated times.
Noteworthy are the lack of adverse signs of toxicity during the
experiment, the very slow clearance with T.sub.1/2>9 hr in each
route and the 80% bioavailability for the compounds administered
IP. Based on these data, we anticipate that 1-3 weekly doses of the
drag I.P in 30 mg/Kg would result in an effective dose for
achieving a constant inhibition of the target.
[0091] FIG. 54. Effects of PDZ1in (113B7) on MDA-9/Syntenin and
c-Src interactions. C8161.9 cells were pre-treated with either DMSO
or a dose of 113B7 (as indicated) and re-plated onto
fibronectin-coated plates. After 30 minutes, cell lysates were
immunoprecipitated and immunoblotted with the indicated antibodies.
IgG, immunoglobulin.
[0092] FIGS. 55A-55D. FIG. 55A. Schematic illustration of the
proposed approach to derive a PDZ focused library against PDZs.
After identification of an initial binding element, a diversity
element scaffold will be identified by the second-site screening
using the SAR by ILOEs approach. Elements of the resulting
bi-dentate libraries will be tested against the given target FIG.
55B. Application of the approach to targeting the PDZ1 domain of
MDA-9/Syntenin, led to compounds 112G4 (K.sub.d.about.300 .mu.M)
and 3D11 (K.sub.d.about.500 .mu.M). Chemical shift mapping studies.
ILOE based second site screening revealed compound 3D11 (K.sub.d in
the millimolar range). Chemical shift mapping data and docked
structure for 3D11 are reported. The bi-dentate compound and its
docked geometry (note that the structure is rotated by 90 degrees
around the horizontal axis) are shown on the right panel. FIG. 55C.
Overlays of HSQC spectra of PDZ12 of MDA-9/Syntenin in presence of
the increasing amounts bi-dentate 113B7 (K.sub.d in low nicromolar
range). FIG. 55D. Synthetic scheme for the the generation of the
PDZ focused library based on the identified scaffolds. The
structure of hit compound 113B7 and titration data are reported
(K.sub.d in low nicromolar range).
[0093] FIG. 56. PCR array for specific adhesion-related molecules.
Aggressive melanoma cell line C8161.9 either expressing control
shRNA or shmda-9 were seeded on fibronectin-coated plates for 6 hr
in growth-starved condition. Total RNA was isolated and subjected
to PCR array according to manufacturer's instruction (SA
Bioscience). Data was analyzed by the software as provided by SA
Bioscience. Select proteins shown.
[0094] FIG. 57. PDZ1in (113B7) treatment results in marked
reduction of B16 experimental lung metastases. C57BU6 mice were
inoculated I.V. with B16 cells (5.times.10) to generate
experimental lung metastases. One day after I.V. injection, mice
received 30 and 50 mg/kg b.w. PDZ1i I.P. 3.times. a week for the
first two weeks (total 6 injections). After 21 days, mice were
sacrificed and lungs were collected, fixed with formalin and
examined for nodules. Representative lungs with tumor metastases
are shown.
[0095] FIG. 58. MDA-9/Syntenin expression facilitates the adhesion
phenotype of cancer cells. MeWO-Luc cells (an aggressive melanoma
cell line that stably expresses luciferase) were pre-infected (in
vitro infection was conducted 48-hr prior to injection) with either
Ad.5/3-null or Ad.5/3-shmda-9 at different m.o.i. and then injected
I.V. into mice. BLI was performed to determine the levels of
circulating metastatic cells in the lungs after 45 min of cell
inoculation.
[0096] FIG. 59. Inhibition of human melanoma metastasis to the
lungs by PDZ1in. MeWo-Luc cells were injected I.V. to establish
experimental lung metastases. Mice received DMSO or drug (3.times.
per week for first two weeks and 2.times./week for next two weeks,
total 8 injections per mice in a 4-week period). BLI images of
whole animals from representative control and experimental groups
are shown.
[0097] FIG. 60. Mice were treated topically with 4-HT and lungs
were collected after 28 days. After confirming the metastatic foci
in lungs (H/E stain, Left panel), sections were subjected to
immunostaining with MDA-9/Syntenin antibody (Right panel).
Representative photo-micrographs are presented.
[0098] FIG. 61. Small molecule PDZ1in (113B7) suppresses melanoma
invasion. MDA-9/Syntenin overexpressed clone of primary immortal
melanocytes (FM-516) and different aggressive melanoma (C8161.9 and
MeWo) cells were pre-treated with either DMSO (vehicle) or compound
at the dose indicated and invasion ability was assayed using a
modified Boyden Chamber according to the manufacturer's
instructions. Photomicrographs were taken at 10.times.
magnification and quantification of the results of three
independent experiments is provided in the graphs+S.D.
DETAILED DESCRIPTION
[0099] MDA-9 is a scaffold protein that plays a key role in tumor
progession and metastasis in cancer. MDA-9 can effect tumor
progession and metastasis through protein-protein interactions. For
example, in breast cancer MDA-9 interacts with TGF.beta.1 to
facilitate epithelial mesenchymal transition (EMT), a key step in
the processes of metastatsis. MDA-9 protein-protein interactions
can occur through binding of a MDA-9 PDZ domain (i.e., PDZ1 domain)
to a downstream target (e.g., TGF.beta.1, c-Src, FAK, STAT3,
IGF-R1, etc.) in the MDA-9 signaling pathway.
[0100] There are an estimated .about.150 PDZ domain-containing
proteins that are involved in cancer and associated with important
physiological processes in transformed cells. Targeting the PDZ
domain to develop specific and effective small molecule inhibitors
has historically proven difficult. However, as described herein,
specific and effective PDZ1 domain binders which target the PDZ1
domain of MDA-9/Syntenin, thereby inhibiting MDA-9 protein-protein
interactions, have been developed for use in cancer treatment.
I. Definitions
[0101] While various embodiments and aspects of the present
invention are shown and described herein, it will be obvious to
those skilled in the art that such embodiments and aspects are
provided by way of example only. Numerous variations, changes, and
substitutions will now occur to those skilled in the art without
departing from the invention. It should be understood that various
alternatives to the embodiments of the invention described herein
may be employed in practicing the invention.
[0102] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
the application including, without limitation, patents, patent
applications, articles, books, manuals, and treatises are hereby
expressly incorporated by reference in their entirety for any
purpose.
[0103] The abbreviations used herein have their conventional
meaning within the chemical and biological arts. The chemical
structures and formulae set forth herein are constructed according
to the standard rules of chemical valency known in the chemical
arts.
[0104] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents that would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is equivalent to --OCH.sub.2--.
[0105] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight (i.e.,
unbranched) or branched carbon chain (or carbon), or combination
thereof, which may be fully saturated, mono- or polyunsaturated and
can include mono-, di- and multivalent radicals. The alkyl may
include a designated number of carbons (e.g., C.sub.1-C.sub.10
means one to ten carbons). Alkyl is an uncyclized chain. Examples
of saturated hydrocarbon radicals include, but are not limited to,
groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An
alkoxy is an alkyl attached to the remainder of the molecule via an
oxygen linker (--O--). An alkyl moiety may be an alkenyl moiety. An
alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully
saturated. An alkenyl may include more than one double bond and/or
one or more triple bonds in addition to the one or more double
bonds. An alkynyl may include more than one triple bond and/or one
or more double bonds in addition to the one or more triple
bonds.
[0106] In embodiments, the term "cycloalkyl" means a monocyclic,
bicyclic, or a multicyclic cycloalkyl ring system. In embodiments,
monocyclic ring systems are cyclic hydrocarbon groups containing
from 3 to 8 carbon atoms, where such groups can be saturated or
unsaturated, but not aromatic. In embodiments, cycloalkyl groups
are fully saturated. Examples of monocyclic cycloalkyls include
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring
systems are bridged monocyclic rings or fused bicyclic rings. In
embodiments, bridged monocyclic rings contain a monocyclic
cycloalkyl ring where two non adjacent carbon atoms of the
monocyclic ring are linked by an alkylene bridge of between one and
three additional carbon atoms (i.e., a bridging group of the form
(CH.sub.2).sub.w, where w is 1, 2, or 3). Representative examples
of bicyclic ring systems include, but are not limited to,
bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane,
bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and
bicyclo[4.2.1]nonane. In embodiments, fused bicyclic cycloalkyl
ring systems contain a monocyclic cycloalkyl ring fused to either a
phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a
monocyclic heterocyclyl, or a monocyclic heteroaryl. In
embodiments, the bridged or fused bicyclic cycloalkyl is attached
to the parent molecular moiety through any carbon atom contained
within the monocyclic cycloalkyl ring. In embodiments, cycloalkyl
groups are optionally substituted with one or two groups which are
independently oxo or thia. In embodiments, the fused bicyclic
cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to
either a phenyl ring, a 3 or 6 membered monocyclic cycloalkyl, a 5
or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic
heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein
the fused bicyclic cycloalkyl is optionally substituted by one or
two groups which are independently oxo or thia. In embodiments,
multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl
ring (base ring) fused to either (i) one ring system selected from
the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a
bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic
heterocyclyl; or (ii) two other ring systems independently selected
from the group consisting of a phenyl, a bicyclic aryl, a
monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic
cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic
or bicyclic heterocyclyl. In embodiments, the multicyclic
cycloalkyl is attached to the parent molecular moiety through any
carbon atom contained within the base ring. In embodiments,
multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl
ring (base ring) fused to either (i) one ring system selected from
the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a
bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic
heterocyclyl; or (ii) two other ring systems independently selected
from the group consisting of a phenyl, a monocyclic heteroaryl, a
monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic
heterocyclyl. Examples of multicyclic cycloalkyl groups include,
but are not limited to tetradecahydrophenanthrenyl,
perhydrophenothiazin-1-yl, and perhydrophenoxazin-1-yl.
[0107] In embodiments, a cycloalkyl is a cycloalkenyl. The term
"cycloalkenyl" is used in accordance with its plain ordinary
meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic,
or a multicyclic cycloalkenyl ring system. In embodiments,
monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups
containing from 3 to 8 carbon atoms, where such groups are
unsaturated (i.e., containing at least one annular carbon carbon
double bond), but not aromatic. Examples of monocyclic cycloalkenyl
ring systems include cyclopentenyl and cyclohexenyl. In
embodiments, bicyclic cycloalkenyl rings are bridged monocyclic
rings or a fused bicyclic rings. In embodiments, bridged monocyclic
rings contain a monocyclic cycloalkenyl ring where two non adjacent
carbon atoms of the monocyclic ring are linked by an alkyl ene
bridge of between one and three additional carbon atoms (i.e., a
bridging group of the form (CH.sub.2).sub.w, where w is 1, 2, or
3). Representative examples of bicyclic cycloalkenyls include, but
are not limited to, norbomenyl and bicyclo[2.2.2]oct 2 enyl. In
embodiments, fused bicyclic cycloalkenyl ring systems contain a
monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic
cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl,
or a monocyclic heteroaryl. In embodiments, the bridged or fused
bicyclic cycloalkenyl is attached to the parent molecular moiety
through any carbon atom contained within the monocyclic
cycloalkenyl ring. In embodiments, cycloalkenyl groups are
optionally substituted with one or two groups which are
independently oxo or thia. In embodiments, multicyclic cycloalkenyl
rings contain a monocyclic cycloalkenyl ring (base ring) fused to
either (i) one ring system selected from the group consisting of a
bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a
bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two
ring systems independently selected from the group consisting of a
phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a
monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic
cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In
embodiments, the multicyclic cycloalkenyl is attached to the parent
molecular moiety through any carbon atom contained within the base
ring. In embodiments, multicyclic cycloalkenyl rings contain a
monocyclic cycloalkenyl ring (base ring) fused to either (i) one
ring system selected from the group consisting of a bicyclic aryl,
a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic
cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems
independently selected from the group consisting of a phenyl, a
monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic
cycloalkenyl, and a monocyclic heterocyclyl.
[0108] In embodiments, a heterocycloalkyl is a heterocyclyl. The
term "heterocyclyl" as used herein, means a monocyclic, bicyclic,
or multicyclic heterocycle. The heterocyclyl monocyclic heterocycle
is a 3, 4, 3, 6 or 7 membered ring containing at least one
heteroatom independently selected from the group consisting of O,
N, and S where the ring is saturated or unsaturated, but not
aromatic. The 3 or 4 membered ring contains 1 heteroatom selected
from the group consisting of O, N and S. The 3 membered ring can
contain zero or one double bond and one, two or three heteroatoms
selected from the group consisting of O, N and S. The 6 or 7
membered ring contains zero, one or two double bonds and one, two
or three heteroatoms selected from the group consisting of O, N and
S. The heterocyclyl monocyclic heterocycle is connected to the
parent molecular moiety through any carbon atom or any nitrogen
atom contained within the heterocyclyl monocyclic heterocycle.
Representative examples of heterocyclyl monocyclic heterocycles
include, but are not limited to, azetidinyl, azepanyl, aziridinyl,
diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,
1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,
isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,
oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl,
piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl,
pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl,
thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl,
thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine
sulfone), thiopyranyl, and trithianyl. The heterocyclyl bicyclic
heterocycle is a monocyclic heterocycle fused to either a phenyl, a
monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic
heterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclic
heterocycle is connected to the parent molecular moiety through any
carbon atom or any nitrogen atom contained within the monocyclic
heterocycle portion of the bicyclic ring system. Representative
examples of bicyclic heterocyclyls include, but are not limited to,
2,3 dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl,
indolin-1-yl, indolin-2-yl, indolin-3-yl,
2,3-dihydrobenzothien-2-yl, decahydroquinolinyl,
decahydroisoquinolinyl, octahydro-1H-indolyl, and
octahydrobenzofuranyl. In embodiments, heterocyclyl groups are
optionally substituted with one or two groups which are
independently oxo or thia. In certain embodiments, the bicyclic
heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring
fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a
5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered
monocyclic heterocyclyl, or a 5 or 6 membered monocyclic
heteroaryl, wherein the bicyclic heterocyclyl is optionally
substituted by one or two groups which are independently oxo or
thia. Multicyclic heterocyclyl ring systems are a monocyclic
heterocyclyl ring (base ring) fused to either (i) one ring system
selected from the group consisting of a bicyclic aryl, a bicyclic
heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a
bicyclic heterocyclyl; or (ii) two other ring systems independently
selected from the group consisting of a phenyl, a bicyclic aryl, a
monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic
cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic
or bicyclic heterocyclyl. The multicyclic heterocyclyl is attached
to the parent molecular moiety through any carbon atom or nitrogen
atom contained within the base ring. In embodiments, multicyclic
heterocyclyl ring systems are a monocyclic heterocyclyl ring (base
ring) fused to either (i) one ring system selected from the group
consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic
cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl;
or (ii) two other ring systems independently selected from the
group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic
cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic
heterocyclyl. Examples of multicyclic heterocyclyl groups include,
but are not limited to 10H-phenothiazin-10-yl,
9,10-dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl,
10H-phenoxazin-10-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl,
1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl,
12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.
[0109] The term "alkylene," by itself or as part of another
substituent, means, unless otherwise stated, a divalent radical
derived from an alkyl, as exemplified, but not limited by,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred herein. A
"lower alkyl" or "lower alkylene" is a shorter chain alkyl or
alkylene group, generally having eight or fewer carbon atoms. The
term "alkenylene," by itself or as part of another substituent,
means, unless otherwise stated, a divalent radical derived from an
alkene.
[0110] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or combinations thereof, including at least one
carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S),
and wherein the nitrogen and sulfur atoms may optionally be
oxidized, and the nitrogen heteroatom may optionally be
quaternized. The heteroatom(s) (e.g., N, S, Si, or P) may be placed
at any interior position of the heteroalkyl group or at the
position at which the alkyl group is attached to the remainder of
the molecule. Heteroalkyl is an uncyclized chain. Examples include,
but are not limited to: --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3,
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3, --O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, and --CN. Up to two or three heteroatoms
may be consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3
and --CH.sub.2--O--Si(CH.sub.3).sub.3. A heteroalkyl moiety may
include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl
moiety may include two optionally different heteroatoms (e.g., O,
N, S, Si, or P). A heteroalkyl moiety may include three optionally
different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl
moiety may include four optionally different heteroatoms (e.g., O,
N, S, Si, or P). A heteroalkyl moiety may include five optionally
different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl
moiety may include up to 8 optionally different heteroatoms (e.g.,
O, N, S, Si, or P). The term "heteroalkenyl," by itself or in
combination with another term, means, unless otherwise stated, a
heteroalkyl including at least one double bond. A heteroalkenyl may
optionally include more than one double bond and/or one or more
triple bonds in additional to the one or more double bonds. The
term "heteroalkynyl," by itself or in combination with another
term, means, unless otherwise stated, a heteroalkyl including at
least one triple bond. A heteroalkynyl may optionally include more
than one triple bond and/or one or more double bonds in additional
to the one or more triple bonds.
[0111] Similarly, the term "heteroalkylene," by itself or as part
of another substituent, means, unless otherwise stated, a divalent
radical derived from heteroalkyl, as exemplified, but not limited
by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--. As described above, heteroalkyl groups, as used
herein, include those groups that are attached to the remainder of
the molecule through a heteroatom, such as --C(O)R', --C(O)NR',
--NR'R'', --OR', --SR', and/or --SO.sub.2R'. Where "heteroalkyl" is
recited, followed by recitations of specific heteroalkyl groups,
such as --NR'R'' or the like, it will be understood that the terms
heteroalkyl and --NR'R'' are not redundant or mutually exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity.
Thus, the term "heteroalkyl" should not be interpreted herein as
excluding specific heteroalkyl groups, such as --NR'R'' or the
like.
[0112] The terms "cycloalkyl" and "heterocycloalkyl," by themselves
or in combination with other terms, mean, unless otherwise stated,
cyclic versions of "alkyl" and "heteroalkyl," respectively.
Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of the molecule. Examples
of cycloalkyl include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl,
3-cyclohexenyl, cycloheptyl, and the like. Examples of
heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a
"heterocycloalkylene," alone or as part of another substituent,
means a divalent radical derived from a cycloalkyl and
heterocycloalkyl, respectively.
[0113] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" includes, but is
not limited to, fluoromethyl, difluoromethyl, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0114] The term "acyl" means, unless otherwise stated, --C(O)R
where R is a substituted or unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0115] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, hydrocarbon substituent, which can be a
single ring or multiple rings (preferably from 1 to 3 rings) that
are fused together (i.e., a fused ring aryl) or linked covalently.
A fused ring aryl refers to multiple rings fused together wherein
at least one of the fused rings is an aryl ring. The term
"heteroaryl" refers to aryl groups (or rings) that contain at least
one heteroatom such as N, O, or S, wherein the nitrogen and sulfur
atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. Thus, the term "heteroaryl" includes fused
ring heteroaryl groups (i.e., multiple rings fused together wherein
at least one of the fused rings is a heteroaromatic ring). A
5,6-fused ring heteroarylene refers to two rings fused together,
wherein one ring has 5 members and the other ring has 6 members,
and wherein at least one ring is a heteroaryl ring. Likewise, a
6,6-fused ring heteroarylene refers to two rings fused together,
wherein one ring has 6 members and the other ring has 6 members,
and wherein at least one ring is a heteroaryl ring. And a 6,5-fused
ring heteroarylene refers to two rings fused together, wherein one
ring has 6 members and the other ring has 5 members, and wherein at
least one ring is a heteroaryl ring. A heteroaryl group can be
attached to the remainder of the molecule through a carbon or
heteroatom. Non-limiting examples of aryl and heteroaryl groups
include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl,
triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl,
isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl,
benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran,
isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl,
quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,
4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. An "arylene" and a "heteroarylene," alone or as
part of another substituent, mean a divalent radical derived from
an aryl and heteroaryl, respectively. A heteroaryl group
substituent may be --O-- bonded to a ring heteroatom nitrogen.
[0116] Spirocyclic rings are two or more rings wherein adjacent
rings are attached through a single atom. The individual rings
within spirocyclic rings may be identical or different Individual
rings in spirocyclic rings may be substituted or unsubstituted and
may have different substituents from other individual rings within
a set of spirocyclic rings. Possible substituents for individual
rings within spirocyclic rings are the possible substituents for
the same ring when not part of spirocyclic rings (e.g. substituents
for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkylene, substituted or unsubstituted
heterocycloalkyl or substituted or unsubstituted
heterocycloalkylene and individual rings within a spirocyclic ring
group may be any of the immediately previous list, including having
all rings of one type (e.g. all rings being substituted
heterocycloalkylene wherein each ring may be the same or different
substituted heterocycloalkylene). When referring to a spirocyclic
ring system, heterocyclic spirocyclic rings means a spirocyclic
rings wherein at least one ring is a heterocyclic ring and wherein
each ring may be a different ring. When referring to a spirocyclic
ring system, substituted spirocyclic rings means that at least one
ring is substituted and each substituent may optionally be
different.
[0117] The symbol "" denotes the point of attachment of a chemical
moiety to the remainder of a molecule or chemical formula.
[0118] The term "oxo," as used herein, means an oxygen that is
double bonded to a carbon atom.
[0119] The term "alkylarylene" as an arylene moiety covalently
bonded to an alkylene moiety (also referred to herein as an
alkylene linker). In embodiments, the alkylarylene group has the
formula:
##STR00003##
[0120] An alkylarylene moiety may be substituted (e.g. with a
substituent group) on the alkylene moiety or the arylene linker
(e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, --N.sub.3,
--CF.sub.3, --CCl.sub.3, --CBr.sub.3, --CI.sub.3, --CN, --CHO,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.2CH.sub.3--SO.sub.3H, --OSO.sub.3H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, substituted or
unsubstituted C.sub.1-C.sub.5 alkyl or substituted or unsubstituted
2 to 5 membered heteroalkyl). In embodiments, the alkylarylene is
unsubstituted.
[0121] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl")
includes both substituted and unsubstituted forms of the indicated
radical. Preferred substituents for each type of radical are
provided below.
[0122] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to,
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --NR'NR''R''', --ONR'R'',
--NR'C(O)NR''NR'''R'''', --CN, --NO.sub.2, --NR'SC.sub.2R'',
--NR'C(O)R'', --NR'C(O)--OR'', --NR'OR'', in a number ranging from
zero to (2m'+1), where m' is the total number of carbon atoms in
such radical. R, R', R'', R''', and R'''' each preferably
independently refer to hydrogen, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl (e.g., aryl substituted with 1-3 halogens), substituted or
unsubstituted heteroaryl, substituted or unsubstituted alkyl,
alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound
described herein includes more than one R group, for example, each
of the R groups is independently selected as are each R', R'',
R''', and R'''' group when more than one of these groups is
present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 4-, 5-, 6-,
or 7-membered ring. For example, --NR'R'' includes, but is not
limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0123] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: --OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'')--NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --NR'NR''R''', --ONR'R'',
--NR'C(O)NR''NR'''R'''', --CN, --NO.sub.2, --R', --N.sub.3,
--CH(Ph).sub.2, fluoro(C.sub.1-C.sub.4)alkoxy, and
fluoro(C.sub.1-C.sub.4)alkyl, --NR'SO.sub.2R'', --NR'C(O)R'',
--NR'C(O)--OR'', --NR'OR'', in a number ranging from zero to the
total number of open valences on the aromatic ring system; and
where R', R'', R''', and R'''' are preferably independently
selected from hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl. When a compound described
herein includes more than one R group, for example, each of the R
groups is independently selected as are each R', R'', R''', and
R'''' groups when more than one of these groups is present.
[0124] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl,
aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene) may be depicted as substituents on the ring rather
than on a specific atom of a ring (commonly referred to as a
floating substituent). In such a case, the substituent may be
attached to any of the ring atoms (obeying the rules of chemical
valency) and in the case of fused rings or spirocyclic rings, a
substituent depicted as associated with one member of the fused
rings or spirocyclic rings (a floating substituent on a single
ring), may be a substituent on any of the fused rings or
spirocyclic rings (a floating substituent on multiple rings). When
a substituent is attached to a ring, but not a specific atom (a
floating substituent), and a subscript for the substituent is an
integer greater than one, the multiple substituents may be on the
same atom, same ring, different atoms, different fused rings,
different spirocyclic rings, and each substituent may optionally be
different. Where a point of attachment of a ring to the remainder
of a molecule is not limited to a single atom (a floating
substituent), the attachment point may be any atom of the ring and
in the case of a fused ring or spirocyclic ring, any atom of any of
the fused rings or spirocyclic rings while obeying the rules of
chemical valency. Where a ring, fused rings, or spirocyclic rings
contain one or more ring heteroatoms and the ring, fused rings, or
spirocyclic rings are shown with one more floating substituents
(including, but not limited to, points of attachment to the
remainder of the molecule), the floating substituents may be bonded
to the heteroatoms. Where the ring heteroatoms are shown bound to
one or more hydrogens (e.g. a ring nitrogen with two bonds to ring
atoms and a third bond to a hydrogen) in the structure or formula
with the floating substituent, when the heteroatom is bonded to the
floating substituent, the substituent will be understood to replace
the hydrogen, while obeying the rules of chemical valency.
[0125] Two or more substituents may optionally be joined to form
aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such
so-called ring-forming substituents are typically, though not
necessarily, found attached to a cyclic base structure. In one
embodiment, the ring-forming substituents are attached to adjacent
members of the base structure. For example, two ring-forming
substituents attached to adjacent members of a cyclic base
structure create a fused ring structure. In another embodiment, the
ring-forming substituents are attached to a single member of the
base structure. For example, two ring-forming substituents attached
to a single member of a cyclic base structure create a spirocyclic
structure. In yet another embodiment, the ring-forming substituents
are attached to non-adjacent members of the base structure.
[0126] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally form a ring of the formula
-T-C(O)--(CRR').sub.q--U--, wherein T and U are independently
--NR--, --O--, --CRR'--, or a single bond, and q is an integer of
from 0 to 3. Alternatively, two of the substituents on adjacent
atoms of the aryl or heteroaryl ring may optionally be replaced
with a substituent of the formula -A-(CH.sub.2).sub.r--B--, wherein
A and B are independently --CRR'--, --O--, --NR--, --S--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2NR'--, or a single bond, and r is an
integer of from 1 to 4. One of the single bonds of the new ring so
formed may optionally be replaced with a double bond.
Alternatively, two of the substituents on adjacent atoms of the
aryl or heteroaryl ring may optionally be replaced with a
substituent of the formula
--(CRR').sub.s--X'--(C''R''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X' is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'', and R''' are preferably independently
selected from hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0127] As used herein, the terms "heteroatom" or "ring heteroatom"
are meant to include oxygen (O), nitrogen (N), sulfur (S),
phosphorus (P), and silicon (Si).
[0128] A "substituent group," as used herein, means a group
selected from the following moieties: [0129] (A) oxo, [0130]
halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH, --S
O.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2, --NHSO.sub.2H,
--NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3, --OCF.sub.3,
--OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2, --OCH Br.sub.2,
--OCHI.sub.2, --OCHF.sub.2, unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4
alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered
heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered
heteroalkyl), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8
cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6
cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl), and [0131] (B) alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least one substituent selected from: [0132] (i)
oxo, [0133] halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3,
--CI.sub.3, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OC Br.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8
membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4
membered heteroalkyl), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl), unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl,
or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl), and [0134] (ii) alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least one substituent selected from: [0135] (a)
oxo, [0136] halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3,
--CI.sub.3, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
.quadrature.NHNH.sub.2, .quadrature.ONH.sub.2,
.quadrature.NHC(O)NHNH.sub.2, .quadrature.NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --O CHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8
membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4
membered heteroalkyl), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl), unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl,
or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl), and [0137] (b) alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least one substituent selected from: oxo,
[0138] halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2, --NHSO.sub.2H,
--NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3, --OCF.sub.3,
--OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2, --OC HBr.sub.2,
--OCHI.sub.2, --OCHF.sub.2, unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4
alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered
heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered
heteroalkyl), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8
cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6
cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl).
[0139] A "size-limited substituent" or "size-limited substituent
group," as used herein, means a group selected from all of the
substituents described above for a "substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl, each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or
unsubstituted aryl is a substituted or unsubstituted
C.sub.6-C.sub.10 aryl, and each substituted or unsubstituted
heteroaryl is a substituted or unsubstituted 5 to 10 membered
heteroaryl.
[0140] A "lower substituent" or "lower substituent group," as used
herein, means a group selected from all of the substituents
described above for a "substituent group," wherein each substituted
or unsubstituted alkyl is a substituted or unsubstituted
C.sub.1-C.sub.8 alkyl, each substituted or unsubstituted
heteroalkyl is a substituted or unsubstituted 2 to 8 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a
substituted or unsubstituted C.sub.3-C.sub.7 cycloalkyl, each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or
unsubstituted aryl is a substituted or unsubstituted
C.sub.6-C.sub.10 aryl, and each substituted or unsubstituted
heteroaryl is a substituted or unsubstituted 5 to 9 membered
heteroaryl.
[0141] In some embodiments, each substituted group described in the
compounds herein is substituted with at least one substituent
group. More specifically, in some embodiments, each substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene described in the
compounds herein are substituted with at least one substituent
group. In other embodiments, at least one or all of these groups
are substituted with at least one size-limited substituent group.
In other embodiments, at least one or all of these groups are
substituted with at least one lower substituent group.
[0142] In other embodiments of the compounds herein, each
substituted or unsubstituted alkyl may be a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl, each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or
unsubstituted aryl is a substituted or unsubstituted
C.sub.6-C.sub.10 aryl, and/or each substituted or unsubstituted
heteroaryl is a substituted or unsubstituted S to 10 membered
heteroaryl. In some embodiments of the compounds herein, each
substituted or unsubstituted alkylene is a substituted or
unsubstituted C.sub.1-C.sub.20 alkylene, each substituted or
unsubstituted heteroalkylene is a substituted or unsubstituted 2 to
20 membered heteroalkylene, each substituted or unsubstituted
cycloalkylene is a substituted or unsubstituted C.sub.3-C.sub.8
cycloalkylene, each substituted or unsubstituted
heterocycloalkylene is a substituted or unsubstituted 3 to 8
membered heterocycloalkylene, each substituted or unsubstituted
arylene is a substituted or unsubstituted C.sub.6-C.sub.10 arylene,
and/or each substituted or unsubstituted heteroarylene is a
substituted or unsubstituted 5 to 10 membered heteroarylene.
[0143] In some embodiments, each substituted or unsubstituted alkyl
is a substituted or unsubstituted C.sub.1-C.sub.8 alkyl, each
substituted or unsubstituted heteroalkyl is a substituted or
unsubstituted 2 to 8 membered heteroalkyl, each substituted or
unsubstituted cycloalkyl is a substituted or unsubstituted
C.sub.3-C.sub.7 cycloalkyl, each substituted or unsubstituted
heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered
heterocycloalkyl, each substituted or unsubstituted aryl is a
substituted or unsubstituted C.sub.6-C.sub.10 aryl, and/or each
substituted or unsubstituted heteroaryl is a substituted or
unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each
substituted or unsubstituted alkylene is a substituted or
unsubstituted C.sub.1-C.sub.8 alkylene, each substituted or
unsubstituted heteroalkylene is a substituted or unsubstituted 2 to
8 membered heteroalkylene, each substituted or unsubstituted
cycloalkylene is a substituted or unsubstituted C.sub.3-C.sub.7
cycloalkylene, each substituted or unsubstituted
heterocycloalkylene is a substituted or unsubstituted 3 to 7
membered heterocycloalkylene, each substituted or unsubstituted
arylene is a substituted or unsubstituted C.sub.6-C.sub.10 arylene,
and/or each substituted or unsubstituted heteroarylene is a
substituted or unsubstituted 5 to 9 membered heteroarylene. In some
embodiments, the compound is a chemical species set forth in the
Examples section, figures, or tables below.
[0144] In embodiments, a substituted or unsubstituted moiety (e.g.,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, and/or substituted or unsubstituted
heteroarylene) is unsubstituted (e.g., is an unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,
unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted
cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted
arylene, and/or unsubstituted heteroarylene, respectively). In
embodiments, a substituted or unsubstituted moiety (e.g.,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, and/or substituted or unsubstituted
heteroarylene) is substituted (e.g., is a substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene, respectively).
[0145] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one substituent group, wherein if the substituted moiety is
substituted with a plurality of substituent groups, each
substituent group may optionally be different. In embodiments, if
the substituted moiety is substituted with a plurality of
substituent groups, each substituent group is different.
[0146] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one size-limited substituent group, wherein if the
substituted moiety is substituted with a plurality of size-limited
substituent groups, each size-limited substituent group may
optionally be different. In embodiments, if the substituted moiety
is substituted with a plurality of size-limited substituent groups,
each size-limited substituent group is different.
[0147] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one lower substituent group, wherein if the substituted
moiety is substituted with a plurality of lower substituent groups,
each lower substituent group may optionally be different. In
embodiments, if the substituted moiety is substituted with a
plurality of lower substituent groups, each lower substituent group
is different.
[0148] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one substituent group, size-limited substituent group, or
lower substituent group; wherein if the substituted moiety is
substituted with a plurality of groups selected from substituent
groups, size-limited substituent groups, and lower substituent
groups; each substituent group, size-limited substituent group,
and/or lower substituent group may optionally be different. In
embodiments, if the substituted moiety is substituted with a
plurality of groups selected from substituent groups, size-limited
substituent groups, and lower substituent groups; each substituent
group, size-limited substituent group, and/or lower substituent
group is different.
[0149] Certain compounds of the present disclosure possess
asymmetric carbon atoms (optical or chiral centers) or double
bonds; the enantiomers, racemates, diastereomers, tautomers,
geometric isomers, stereoisomeric forms that may be defined, in
terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or
(L)- for amino acids, and individual isomers are encompassed within
the scope of the present disclosure. The compounds of the present
disclosure do not include those that are known in art to be too
unstable to synthesize and/or isolate. The present disclosure is
meant to include compounds in racemic and optically pure forms.
Optically active (R)- and (S)-, or (D)- and (L)-isomers may be
prepared using chiral synthons or chiral reagents, or resolved
using conventional techniques. When the compounds described herein
contain olefinic bonds or other centers of geometric asymmetry, and
unless specified otherwise, it is intended that the compounds
include both E and Z geometric isomers.
[0150] As used herein, the term "isomers" refers to compounds
having the same number and kind of atoms, and hence the same
molecular weight, but differing in respect to the structural
arrangement or configuration of the atoms.
[0151] The term "tautomer," as used herein, refers to one of two or
more structural isomers which exist in equilibrium and which are
readily converted from one isomeric form to another. For example,
formula I may be written as
##STR00004##
Additionally, any formula or compound described herein may be
written as either isomeric (e.g. tautomeric) form.
[0152] It will be apparent to one skilled in the art that certain
compounds of this disclosure may exist in tautomeric forms, all
such tautomeric forms of the compounds being within the scope of
the disclosure.
[0153] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the disclosure.
[0154] Unless otherwise stated, structures depicted herein are also
meant to include compounds which differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of a hydrogen by
a deuterium or tritium, or the replacement of a carbon by .sup.13C-
or .sup.14C-enriched carbon are within the scope of this
disclosure.
[0155] The compounds of the present disclosure may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I), or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present disclosure,
whether radioactive or not, are encompassed within the scope of the
present disclosure.
[0156] It should be noted that throughout the application that
alternatives are written in Markush groups, for example, each amino
acid position that contains more than one possible amino acid. It
is specifically contemplated that each member of the Markush group
should be considered separately, thereby comprising another
embodiment, and the Markush group is not to be read as a single
unit.
[0157] "Analog," or "analogue" is used in accordance with its plain
ordinary meaning within Chemistry and Biology and refers to a
chemical compound that is structurally similar to another compound
(i.e., a so-called "reference" compound) but differs in
composition, e.g., in the replacement of one atom by an atom of a
different element, or in the presence of a particular functional
group, or the replacement of one functional group by another
functional group, or the absolute stereochemistry of one or more
chiral centers of the reference compound. Accordingly, an analog is
a compound that is similar or comparable in function and appearance
but not in structure or origin to a reference compound.
[0158] The terms "a" or "an," as used in herein means one or more.
In addition, the phrase "substituted with a[n]," as used herein,
means the specified group may be substituted with one or more of
any or all of the named substituents. For example, where a group,
such as an alkyl or heteroaryl group, is "substituted with an
unsubstituted C.sub.1-C.sub.20 alkyl, or unsubstituted 2 to 20
membered heteroalkyl," the group may contain one or more
unsubstituted C.sub.1-C.sub.20 alkyls, and/or one or more
unsubstituted 2 to 20 membered heteroalkyls.
[0159] Moreover, where a moiety is substituted with an R
substituent, the group may be referred to as "R-substituted." Where
a moiety is R-substituted, the moiety is substituted with at least
one R substituent and each R substituent is optionally different
Where a particular R group is present in the description of a
chemical genus (such as Formula (I)), a Roman alphabetic symbol may
be used to distinguish each appearance of that particular R group.
For example, where multiple R.sup.13 substituents are present, each
R.sup.13 substituent may be distinguished as R.sup.13A, R.sup.13B,
R.sup.13C, R.sup.13D, etc., wherein each of R.sup.13A, R.sup.13B,
R.sup.13C, R.sup.13D, etc. is defined within the scope of the
definition of R.sup.13 and optionally differently.
[0160] Descriptions of compounds of the present disclosure are
limited by principles of chemical bonding known to those skilled in
the art. Accordingly, where a group may be substituted by one or
more of a number of substituents, such substitutions are selected
so as to comply with principles of chemical bonding and to give
compounds which are not inherently unstable and/or would be known
to one of ordinary skill in the art as likely to be unstable under
ambient conditions, such as aqueous, neutral, and several known
physiological conditions. For example, a heterocycloalkyl or
heteroaryl is attached to the remainder of the molecule via a ring
heteroatom in compliance with principles of chemical bonding known
to those skilled in the art thereby avoiding inherently unstable
compounds.
[0161] As used herein, the term "about" means a range of values
including the specified value, which a person of ordinary skill in
the art would consider reasonably similar to the specified value.
In embodiments, the term "about" means within a standard deviation
using measurements generally acceptable in the art. In embodiments,
about means a range extending to +/-10% of the specified value. In
embodiments, about means the specified value.
[0162] The terms "a" or "an," as used in herein means one or more.
In addition, the phrase "substituted with a[n]," as used herein,
means the specified group may be substituted with one or more of
any or all of the named substituents. For example, where a group,
such as an alkyl or heteroaryl group, is "substituted with an
unsubstituted C.sub.1-C.sub.20 alkyl, or unsubstituted 2 to 20
membered heteroalkyl," the group may contain one or more
unsubstituted C.sub.1-C.sub.20 alkyls, and/or one or more
unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a
moiety is substituted with an R substituent, the group may be
referred to as "R-substituted." Where a moiety is R-substituted,
the moiety is substituted with at least one R substituent and each
R substituent is optionally different.
[0163] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by a
person of ordinary skill in the art. See, e.g., Singleton et al,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley
& Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR
CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold
Springs Harbor, N Y 1989). Any methods, devices and materials
similar or equivalent to those described herein can be used in the
practice of this invention. The following definitions are provided
to facilitate understanding of certain terms used frequently herein
and are not meant to limit the scope of the present disclosure.
[0164] As may be used herein, the terms "nucleic acid," "nucleic
acid molecule," "nucleic acid oligomer," "oligonucleotide,"
"nucleic acid sequence," "nucleic acid fragment" and
"polynucleotide" are used interchangeably and are intended to
include, but are not limited to, a polymeric form of nucleotides
covalently linked together that may have various lengths, either
deoxyribonucleotides or ribonucleotides, or analogs, derivatives or
modifications thereof. Different polynucleotides may have different
three-dimensional structures, and may perform various functions,
known or unknown. Non-limiting examples of polynucleotides include
a gene, a gene fragment, an exon, an intron, intergenic DNA
(including, without limitation, heterochromatic DNA), messenger RNA
(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a
recombinant polynucleotide, a branched polynucleotide, a plasmid, a
vector, isolated DNA of a sequence, isolated RNA of a sequence, a
nucleic acid probe, and a primer. Polynucleotides useful in the
methods of the disclosure may comprise natural nucleic acid
sequences and variants thereof, artificial nucleic acid sequences,
or a combination of such sequences.
[0165] A polynucleotide is typically composed of a specific
sequence of four nucleotide bases: adenine (A); cytosine (C);
guanine (G); and thymine (T) (uracil (U) for thymine (T) when the
polynucleotide is RNA). Thus, the term "polynucleotide sequence" is
the alphabetical representation of a polynucleotide molecule;
alternatively, the term may be applied to the polynucleotide
molecule itself. This alphabetical representation can be input into
databases in a computer having a central processing unit and used
for bioinformatics applications such as functional genomics and
homology searching. Polynucleotides may optionally include one or
more non-standard nucleotide(s), nucleotide analog(s) and/or
modified nucleotides.
[0166] The term "phosphorothioate nucleic acid" refers to a nucleic
acid in which one or more internucleotide linkages are through a
phosphorothioate moiety (thiophosphate) moiety. The
phosphorothioate moiety may be a monothiophosphate
(--P(O).sub.3(S).sup.3---) or a dithiophosphate
(--P(O).sub.2(S).sub.2.sup.3---). In embodiments of all the aspects
provided herein, the phosphorothioate moiety is a monothiophosphate
(--P(O).sub.3(S).sup.3---). That is, in embodiments of all the
aspects provided herein, the phosphorothioate nucleic acid is a
monothiophosphate nucleic acid. In embodiments, one or more of the
nucleosides of a phosphorothioate nucleic acid are linked through a
phosphorothioate moiety (e.g. monothiophosphate) moiety, and the
remaining nucleosides are linked through a phosphodiester moiety
(--P(O).sub.4.sup.3---). In embodiments, one or more of the
nucleosides of a phosphorothioate nucleic acid are linked through a
phosphorothioate moiety (e.g. monothiophosphate) moiety, and the
remaining nucleosides are linked through a methylphosphonate
linkage. In embodiments, all the nucleosides of a phosphorothioate
nucleic acid are linked through a phosphorothioate moiety (e.g. a
monothiophosphate) moiety.
[0167] Phosphorothioate oligonucleotides (phosphorothioate nucleic
acids) are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30,
40, 50 or more nucleotides in length, up to about 100 nucleotides
in length. Phosphorothioate nucleic acids may also be longer in
lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000,
etc. As described above, in certain embodiments, the
phosphorothioate nucleic acids herein contain one or more
phosphodiester bonds. In other embodiments, the phosphorothioate
nucleic acids include alternate backbones (e.g., mimics or analogs
of phosphodiesters as known in the art, such as, boranophosphate,
methylphosphonate, phosphoramidate, or O-methylphophoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press). The phosphorothioate nucleic
acids may also include one or more nucleic acid analog monomers
known in the art, such as, peptide nucleic acid monomer or polymer,
locked nucleic acid monomer or polymer, morpholino monomer or
polymer, glycol nucleic acid monomer or polymer, or threose nucleic
acid monomer or polymer. Other analog nucleic acids include those
with positive backbones; non-ionic backbones, and nonribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
Carbohydrate Modifications in Antisense Research, Sanghui &
Cook, eds. Nucleic acids containing one or more carbocyclic sugars
are also included within one definition of nucleic acids.
Modifications of the ribose-phosphate backbone may be done for a
variety of reasons, e.g., to increase the stability and half-life
of such molecules in physiological environments or as probes on a
biochip. Mixtures of naturally occurring nucleic acids and analogs
can be made; alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made. Phosphorothioate nucleic acids and
phosphorothioate polymer backbones can be linear or branched. For
example, the branched nucleic acids are repetitively branched to
form higher ordered structures such as dendrimers and the like.
[0168] As used herein, a "phosphorothioate polymer backbone" is a
chemical polymer with at least two phosphorothioate linkages (e.g.
monothiophosphate) (e.g. linking together sugar subunits, cyclic
subunits or alkyl subunits). The phosphorothioate polymer backbone
may be a phosphorothioate sugar polymer, which is a
phosphorothioate nucleic acid in which one or more (or all) of the
chain of pentose sugars lack the bases (nucleobases) normally
present in a nucleic acid. The phosphorothioate polymer backbone
can include two or more phosphorothioate linkages. The
phosphorothioate polymer backbone can include 5, 6, 1, 8, 9, 10,
12, 15, 25, 30, 40, 50 or more linkages and can contain up to about
100 phosphorothioate linkages. Phosphorothioate polymer backbones
may also contain a larger number of linkages, e.g., 200, 300, 500,
1000, 2000, 3000, 5000, 7000, 10,000, and the like.
[0169] The phosphorothioate nucleic acids and phosphorothioate
polymer backbones may be partially or completely phosphorothioated.
For example, 50% or more of the internucleotide linkages of a
phosphorothioate nucleic acid can be phosphorothioate linkages.
Optionally, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the
internucleotide linkages of a phosphorothioate nucleic acid are
phosphorothioate linkages. Optionally, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% of the internucleotide linkages of
a phosphorothioate nucleic acid are phosphorothioate linkages.
Optionally, 75%, 80%, 85%, 90%, 95%, or 99% of the internucleotide
linkages of a phosphorothioate nucleic acid are phosphorothioate
linkages. Optionally, 90%, 95%, or 99% of the internucleotide
linkages of a phosphorothioate nucleic acid are phosphorothioate
linkages. In embodiments, the remaining internucleotide linkages
are phosphodiester linkages. In embodiments, the remaining
internucleotide linkages are methylphosphonate linkages.
Optionally, 100% of the internucleotide linkages of the
phosphorothioate nucleic acids are phosphorothioate linkages.
Similarly, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar
linkages in a phosphorothioate polymer backbone can be
phosphorothioate linkages. Optionally, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar linkages in a
phosphorothioate polymer backbone can be phosphorothioate linkages.
Optionally, 75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar
linkages in a phosphorothioate polymer backbone can be
phosphorothioate linkages. Optionally, 90%, 95%, or 99%, of the
intersugar linkages in a phosphorothioate polymer backbone can be
phosphorothioate linkages. In embodiments, the remaining
internucleotide linkages are phosphodiester linkages. In
embodiments, the remaining internucleotide linkages are
methylphosphonate linkages. Optionally, 100% of the intersugar
linkages of the phosphorothioate polymer backbone are
phosphorothioate linkages.
[0170] Optionally, about 5%, about 10%, about 15%, about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 95%, or about 99% of the internucleotide
linkages of a phosphorothioate nucleic acid are phosphorothioate
linkages. Optionally, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
or about 99% of the internucleotide linkages of a phosphorothioate
nucleic acid are phosphorothioate linkages. Optionally, about 75%,
about 80%, about 85%, about 90%, about 95%, or about 99% of the
internucleotide linkages of a phosphorothioate nucleic acid are
phosphorothioate linkages. Optionally, about 90%, about 95%, or
about 99% of the internucleotide linkages of a phosphorothioate
nucleic acid are phosphorothioate linkages. In embodiments, the
remaining internucleotide linkages are phosphodiester linkages. In
embodiments, the remaining internucleotide linkages are
methylphosphonate linkages. Optionally, about 100% of the
internucleotide linkages of the phosphorothioate nucleic acids are
phosphorothioate linkages. Similarly, about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95%, or about 99%, of
the intersugar linkages in a phosphorothioate polymer backbone can
be phosphorothioate linkages. Optionally, about 50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about 95%, or about 99%, of the intersugar linkages in a
phosphorothioate polymer backbone can be phosphorothioate linkages.
Optionally, about 75%, about 80%, about 85%, about 90%, about 95%,
or about 99%, of the intersugar linkages in a phosphorothioate
polymer backbone can be phosphorothioate linkages. Optionally,
about 90%, about 95%, or about 99%, of the intersugar linkages in a
phosphorothioate polymer backbone can be phosphorothioate linkages.
In embodiments, the remaining internucleotide linkages are
phosphodiester linkages. In embodiments, the remaining
internucleotide linkages are methylphosphonate linkages.
Optionally, about 100% of the intersugar linkages of the
phosphorothioate polymer backbone are phosphorothioate
linkages.
[0171] The term "aptamer" as provided herein refers to
oligonucleotides (e.g. short oligonucleotides or
deoxyribonucleotides), that bind (e.g. with high affinity and
specificity) to proteins, peptides, and small molecules. Aptamers
may have secondary or tertiary structure and, thus, may be able to
fold into diverse and intricate molecular structures. Aptamers can
be selected in vitro from very large libraries of randomized
sequences by the process of systemic evolution of ligands by
exponential enrichment (SELEX as described in Ellington A D,
Szostak J W (1990) In vitro selection of RNA molecules that bind
specific ligands. Nature 346:818-822; Tuerk C, Gold L (1990)
Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase. Science 249:505-510) or
by developing SOMAmers (slow off-rate modified aptamers) (Gold L et
al. (2010) Aptamer-based multiplexed proteomic technology for
biomarker discovery. PLoS ONE 5(12):el 5004). Applying the SELEX
and the SOMAmer technology includes for instance adding functional
groups that mimic amino acid side chains to expand the aptamer's
chemical diversity. As a result high affinity aptamers for almost
any protein target are enriched and identified. Aptamers exhibit
many desirable properties for targeted drug delivery, such as ease
of selection and synthesis, high binding affinity and specificity,
low immunogenicity, and versatile synthetic accessibility. To date,
a variety of anti-cancer agents (e.g. chemotherapy drugs, toxins,
and siRNAs) have been successfully delivered to cancer cells in
vitro using apatmers.
[0172] The word "expression" or "expressed" as used herein in
reference to a gene means the transcriptional and/or translational
product of that gene. The level of expression of a DNA molecule in
a cell may be determined on the basis of either the amount of
corresponding mRNA that is present within the cell or the amount of
protein encoded by that DNA produced by the cell. The level of
expression of non-coding nucleic acid molecules (e.g., microRNA)
may be detected by standard PCR or Northern blot methods well known
in the art See, Sambrook et al., 1989 Molecular Clotting: A
Laboratory Manual, 18.1-18.88.
[0173] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid. The terms
"non-naturally occurring amino acid" and "unnatural amino acid"
refer to amino acid analogs, synthetic amino acids, and amino acid
mimetics which are not found in nature.
[0174] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0175] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues, wherein the polymer may In embodiments be conjugated to a
moiety that does not consist of amino acids. The terms apply to
amino acid polymers in which one or more amino acid residue is an
artificial chemical mimetic of a corresponding naturally occurring
amino acid, as well as to naturally occurring amino acid polymers
and non-naturally occurring amino acid polymers. A "fusion protein"
refers to a chimeric protein encoding two or more separate protein
sequences that are recombinantly expressed as a single moiety.
[0176] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids that encode identical or essentially identical amino
acid sequences. Because of the degeneracy of the genetic code, a
number of nucleic acid sequences will encode any given protein. For
instance, the codons GCA, GCC, GCG and GCU all encode the amino
acid alanine. Thus, at every position where an alanine is specified
by a codon, the codon can be altered to any of the corresponding
codons described without altering the encoded polypeptide. Such
nucleic acid variations are "silent variations," which are one
species of conservatively modified variations. Every nucleic acid
sequence herein which encodes a polypeptide also describes every
possible silent variation of the nucleic acid. One of skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid which encodes a polypeptide is implicit
in each described sequence.
[0177] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the disclosure.
[0178] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0179] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (Q, Methionine (M)
[0180] (see, e.g., Creighton, Proteins (1984)).
[0181] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide or polypeptide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0182] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site
http://www.ncbijilm.nih.gov/BLAST/ or the like). Such sequences are
then said to be "substantially identical." This definition also
refers to, or may be applied to, the compliment of a test sequence.
The definition also includes sequences that have deletions and/or
additions, as well as those that have substitutions. As described
below, the preferred algorithms can account for gaps and the like.
Preferably, identity exists over a region that is at least about 25
amino acids or nucleotides in length, or more preferably over a
region that is 50-100 amino acids or nucleotides in length.
[0183] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0184] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of, e.g., a full length sequence or from
20 to 600, about 50 to about 200, or about 100 to about 150 amino
acids or nucleotides in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are well known in the art. Optimal
alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith and Waterman (1970) Adv.
Appl. Math. 2:482c, by the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for
similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad.
Sci. USA 85:2444, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0185] An example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbijilm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) or 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands.
[0186] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0187] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross-reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0188] An amino acid or nucleotide base "position" is denoted by a
number that sequentially identifies each amino acid (or nucleotide
base) in the reference sequence based on its position relative to
the N-terminus (or 5'-end). Due to deletions, insertions,
truncations, fusions, and the like that must be taken into account
when determining an optimal alignment, in general the amino acid
residue number in a test sequence determined by simply counting
from the N-terminus will not necessarily be the same as the number
of its corresponding position in the reference sequence. For
example, in a case where a variant has a deletion relative to an
aligned reference sequence, there will be no amino acid in the
variant that corresponds to a position in the reference sequence at
the site of deletion. Where there is an insertion in an aligned
reference sequence, that insertion will not correspond to a
numbered amino acid position in the reference sequence. In the case
of truncations or fusions there can be stretches of amino acids in
either the reference or aligned sequence that do not correspond to
any amino acid in the corresponding sequence.
[0189] The terms "numbered with reference to" or "corresponding
to," when used in the context of the numbering of a given amino
acid or polynucleotide sequence, refers to the numbering of the
residues of a specified reference sequence when the given amino
acid or polynucleotide sequence is compared to the reference
sequence.
[0190] An amino acid residue in a protein "corresponds" to a given
residue when it occupies the same essential structural position
within the protein as the given residue. For example, a selected
residue in a selected protein corresponds to, for example,
glutamine at position 110 of a human MDA-9 protein when the
selected residue occupies the same essential spatial or other
structural relationship as a glutamine at position 110 in human
MDA-9 protein. In some embodiments, where a selected protein is
aligned for maximum homology with the human MDA-9 protein, the
position in the aligned selected protein aligning with glutamine
110 is said to correspond to glutamine 110. Instead of a primary
sequence alignment, a three dimensional structural alignment can
also be used, e.g., where the structure of the selected protein is
aligned for maximum correspondence with the human MDA-9 protein and
the overall structures compared. In this case, an amino acid that
occupies the same essential position as glutamine 110 in the
structural model is said to correspond to the glutamine 110
residue.
[0191] A "cell" as used herein, refers to a cell carrying out
metabolic or other functions sufficient to preserve or replicate
its genomic DNA. A cell can be identified by well-known methods in
the art including, for example, presence of an intact membrane,
staining by a particular dye, ability to produce progeny or, in the
case of a gamete, ability to combine with a second gamete to
produce a viable offspring. Cells may include prokaryotic and
eukaryotic cells. Prokaryotic cells include but are not limited to
bacteria. Eukaryotic cells include but are not limited to yeast
cells and cells derived from plants and animals, for example
mammalian, insect (e.g., spodoptera) and human cells. Cells may be
useful when they are naturally nonadherent or have been treated not
to adhere to surfaces, for example by trypsinization.
[0192] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody plays a
significant role in determining the specificity and affinity of
binding. In some embodiments, antibodies or fragments of antibodies
may be derived from different organisms, including humans, mice,
rats, hamsters, camels, etc. Antibodies of the invention may
include antibodies that have been modified or mutated at one or
more amino acid positions to improve or modulate a desired function
of the antibody (e.g. glycosylation, expression, antigen
recognition, effector functions, antigen binding, specificity,
etc.).
[0193] Antibodies are large, complex molecules (molecular weight of
.about.150,000 or about 1320 amino acids) with intricate internal
structure. A natural antibody molecule contains two identical pairs
of polypeptide chains, each pair having one light chain and one
heavy chain. Each light chain and heavy chain in turn consists of
two regions: a variable ("V") region involved in binding the target
antigen, and a constant ("C") region that interacts with other
components of the immune system. The light and heavy chain variable
regions come together in 3-dimensional space to form a variable
region that binds the antigen (for example, a receptor on the
surface of a cell). Within each light or heavy chain variable
region, there are three short segments (averaging 10 amino acids in
length) called the complementarity determining regions ("CDRs").
The six CDRs in an antibody variable domain (three from the light
chain and three from the heavy chain) fold up together in
3-dimensional space to form the actual antibody binding site which
docks onto the target antigen. The position and length of the CDRs
have been precisely defined by Kabat, E. et al., Sequences of
Proteins of Immunological Interest, U.S. Department of Health and
Human Services, 1983, 1987. The part of a variable region not
contained in the CDRs is called the framework ("FR"), which forms
the environment for the CDRs.
[0194] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (VL) or light chain variable region and
variable heavy chain (VH) or heavy chain variable region refer to
these light and heavy chain regions, respectively. The terms
variable light chain (VL) and light chain variable region as
referred to herein may be used interchangeably. The terms variable
heavy chain (VH) and heavy chain variable region as referred to
herein may be used interchangeably. The Fc (i.e. fragment
crystallizable region) is the "base" or "tail" of an immunoglobulin
and is typically composed of two heavy chains that contribute two
or three constant domains depending on the class of the antibody.
By binding to specific proteins the Fc region ensures that each
antibody generates an appropriate immune response for a given
antigen. The Fc region also binds to various cell receptors, such
as Fc receptors, and other immune molecules, such as complement
proteins.
[0195] The term "antigen" as provided herein refers to molecules
capable of binding to the antibody region provided herein, wherein
the binding site is not the peptide binding site.
[0196] Antibodies exist, for example, as intact immunoglobulins or
as a number of well-characterized fragments produced by digestion
with various peptidases. Thus, for example, pepsin digests an
antibody below the disulfide linkages in the hinge region to
produce F(ab)'2, a dimer of Fab which itself is a light chain
joined to VH--CH1 by a disulfide bond. The F(ab)'2 may be reduced
under mild conditions to break the disulfide linkage in the hinge
region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
The Fab' monomer is essentially the antigen binding portion with
part of the hinge region (see Fundamental Immunology (Paul ed., 3d
ed. 1993). While various antibody fragments are defined in terms of
the digestion of an intact antibody, one of skill will appreciate
that such fragments may be synthesized de novo either chemically or
by using recombinant DNA methodology. Thus, the term antibody, as
used herein, also includes antibody fragments either produced by
the modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990)).
[0197] A single-chain variable fragment (scFv) is typically a
fusion protein of the variable regions of the heavy (VH) and light
chains (VL) of immunoglobulins, connected with a short linker
peptide of 10 to about 25 amino acids. The linker may usually be
rich in glycine for flexibility, as well as serine or threonine for
solubility. The linker can either connect the N-terminus of the VH
with the C-terminus of the VL, or vice versa.
[0198] The epitope of a mAb is the region of its antigen to which
the mAb binds. Two antibodies bind to the same or overlapping
epitope if each competitively inhibits (blocks) binding of the
other to the antigen. That is, a 1.times., 5.times., 10.times.,
20.times. or 100.times. excess of one antibody inhibits binding of
the other by at least 30% but preferably 50%, 75%, 90% or even 99%
as measured in a competitive binding assay (see, e.g., Junghans et
al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have
the same epitope if essentially all amino acid mutations in the
antigen that reduce or eliminate binding of one antibody reduce or
eliminate binding of the other. Two antibodies have overlapping
epitopes if some amino acid mutations that reduce or eliminate
binding of one antibody reduce or eliminate binding of the
other.
[0199] For preparation of suitable antibodies of the invention and
for use according to the invention, e.g., recombinant, monoclonal,
or polyclonal antibodies, many techniques known in the art can be
used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975);
Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-%
in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
(1985); Coligan, Current Protocols in Immunology (1991); Harlow
& Lane, Antibodies, A Laboratory Manual (1988); and Goding,
Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The
genes encoding the heavy and light chains of an antibody of
interest can be cloned from a cell, e.g., the genes encoding a
monoclonal antibody can be cloned from a hybridoma and used to
produce a recombinant monoclonal antibody. Gene libraries encoding
heavy and light chains of monoclonal antibodies can also be made
from hybridoma or plasma cells. Random combinations of the heavy
and light chain gene products generate a large pool of antibodies
with different antigenic specificity (see, e.g., Kuby, Immunology
(3rd ed. 1997)). Techniques for the production of single chain
antibodies or recombinant antibodies (U.S. Pat. Nos. 4,946,778,
4,816,567) can be adapted to produce antibodies to polypeptides of
this invention. Also, transgenic mice, or other organisms such as
other mammals, may be used to express humanized or human antibodies
(see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology
10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994);
Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature
Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology
14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol.
13:65-93 (1995)). Alternatively, phage display technology can be
used to identify antibodies and heteromeric Fab fragments that
specifically bind to selected antigens (see, e.g., McCafferty et
al., Nature 348:552-554 (1990); Marks et al., Biotechnology
10:779-783 (1992)). Antibodies can also be made bispecific, i.e.,
able to recognize two different antigens (see, e.g., WO 93/08829,
Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al.,
Methods in Enzymology 121:210 (1986)). Antibodies can also be
heteroconjugates, e.g., two covalently joined antibodies, or
immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO
92/200373; and EP 03089).
[0200] Methods for humanizing or primatizing non-human antibodies
are well known in the art (e.g., U.S. Pat. Nos. 4,816,567;
5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085;
6,180,370; 6,210,671; and 6,329,511; WO 87/02671; EP Patent
Application 0173494; Jones et al. (1986) Nature 321:522; and
Verhoyen et al. (1988) Science 239:1534). Humanized antibodies are
further described in, e.g., Winter and Milstein (1991) Nature
349:293. Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers (see, e.g., Morrison et al., PNAS USA,
81:6851-6855 (1984), Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv.
Immunol., 44:65-92 (1988), Verhoeyen et al., Science 239:1534-1536
(1988) and Presta, Curr. Op. Struct Biol. 2:593-596 (1992), Padlan,
Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,
31(3):169-217 (1994)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. Accordingly,
such humanized antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies. For example, polynucleotides comprising a
first sequence coding for humanized immunoglobulin framework
regions and a second sequence set coding for the desired
immunoglobulin complementarity determining regions can be produced
synthetically or by combining appropriate cDNA and genomic DNA
segments. Human constant region DNA sequences can be isolated in
accordance with well known procedures from a variety of human
cells.
[0201] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity. The preferred antibodies of and for use
according to the invention include humanized and/or chimeric
monoclonal antibodies.
[0202] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologies. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
typically requires an antibody that is selected for its specificity
for a particular protein. For example, polyclonal antibodies can be
selected to obtain only a subset of antibodies that are
specifically immunoreactive with the selected antigen and not with
other proteins. This selection may be achieved by subtracting out
antibodies that cross-react with other molecules. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Using Antibodies, A Laboratory Manual (1998) for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity).
[0203] A "ligand" refers to an agent, e.g., a polypeptide or other
molecule, capable of binding to a receptor or target polypeptide
(e.g., PDZ1 domain).
[0204] The term "isolated", when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
See of other cellular components with which it is associated in the
natural state. It can be, for example, in a homogeneous state and
may be in either a dry or aqueous solution. Purity and homogeneity
are typically determined using analytical chemistry techniques such
as polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein that is the predominant species present
in a preparation is substantially purified.
[0205] "Contacting" is used in accordance with its plain ordinary
meaning and refers to the process of allowing at least two distinct
species (e.g. chemical compounds including biomolecules or cells)
to become sufficiently proximal to react, interact or physically
touch. It should be appreciated; however, the resulting reaction
product can be produced directly from a reaction between the added
reagents or from an intermediate from one or more of the added
reagents which can be produced in the reaction mixture.
[0206] The term "contacting" may include allowing two species to
react, interact, or physically touch, wherein the two species may
be, for example, a PDZ1 domain binder (e.g., small molecule,
antibody, aptamer, ligand, or compound as described herein and a
polypetide provided herein (e.g., MDA-9 (e.g., PDZ1 domain)). In
embodiments, contacting includes, for example, allowing a PDZ1
domain binder as described herein, including embodiments thereof,
to interact with a PDZ1 domain of a MDA-9 protein.
[0207] As defined herein, the term "inhibition", "inhibit",
"inhibiting" and the like in reference to a protein-binder (e.g.,
PDZ1 domain binder) interaction means negatively affecting (e.g.
decreasing) the activity or function of the protein (e.g., MDA-9)
relative to the activity or function of the protein in the absence
of the binder. In embodiments, inhibition means negatively
affecting (e.g. decreasing) the concentration or levels of the
protein relative to the concentration or level of the protein in
the absence of the binder. In embodiments, inhibition refers to
reduction of a disease or symptoms of disease. In embodiments,
inhibition refers to a reduction in the activity of a particular
protein target Thus, inhibition includes, at least in part,
partially or totally blocking stimulation, decreasing, preventing,
or delaying activation, or inactivating, desensitizing, or
down-regulating signal transduction or enzymatic activity or the
amount of a protein. In embodiments, inhibition refers to a
reduction of activity of a target protein resulting from a direct
interaction (e.g. an inhibitor binds to the target protein). In
embodiments, inhibition refers to a reduction of activity of a
target protein from an indirect interaction (e.g. an inhibitor
binds to a protein that activates the target protein, thereby
preventing target protein activation). A "PDZ1 domain binder" is a
compound that negatively affects (e.g. decreases) the activity or
function of MDA-9 relative to the activity or function of MDA-9 in
the absence of the PDZ1 domain binder by binding to the PDZ1 domain
of the MDA-9 protein. A decrease in MDA-9 activity may result in
changes in the signaling pathway downstream of MDA-9. For example,
MDA-9 inhibition by a PDZ1 domain binder may decrease activation,
activity, expression, or stability of TGF.beta.1, p38 MAPK,
NF-.kappa.B, STAT3, IGF-R1, AEG-1, JNK, EGFR, AKT, phohoinositide
3-kinase, FAK, and/or c-Src.
[0208] The term "signaling pathway" as used herein refers to a
series of interactions between cellular and optionally
extra-cellular components (e.g. proteins, nucleic acids, small
molecules, ions, lipids) that conveys a change in one component to
one or more other components, which in turn may convey a change to
additional components, which is optionally propagated to other
signaling pathway components. For example, binding of a MDA-9
protein with a compound as described herein (e.g., PDZ1 domain
binder) may reduce the level of a product of the MDA-9 catalyzed
reaction or the level of a downstream derivatives of the product or
binding may reduce the interactions between MDA-9 or an MDA-9
reaction product and downstream effectors or signaling pathway
components, resulting in changes in cell growth, proliferation,
metastasis, or survival.
[0209] As defined herein, the term "activation", "activate",
"activating" and the like in reference to a protein refers to
conversion of a protein into a biologically active derivative from
an initial inactive or deactivated state. The terms reference
activation, or activating, sensitizing, or up-regulating signal
transduction or enzymatic activity or the amount of a protein
decreased in a disease.
[0210] The terms "agonist," "activator," "upregulator," etc. refer
to a substance capable of detectably increasing the expression or
activity of a given gene or protein. The agonist can increase
expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or more in comparison to a control in the absence of the agonist.
In certain instances, expression or activity is 1.5-fold, 2-fold,
3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or
activity in the absence of the agonist.
[0211] The terms "inhibitor," "repressor" or "antagonist" or
"downregulator" interchangeably refer to a substance capable of
detectably decreasing the expression or activity of a given gene or
protein. The antagonist can decrease expression or activity 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a
control in the absence of the antagonist. In certain instances,
expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,
10-fold or lower than the expression or activity in the absence of
the antagonist.
[0212] The terms "MDA-9," "Syntenin," "MDA-9/Syntenin," or "SDCBP"
refer to a protein (including homologs, isoforms, and functional
fragments thereof) with MDA-9 activity. The term includes any
recombinant or naturally-occurring form of MDA-9 variants thereof
that maintain MDA-9 activity (e.g. within at least 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or 100% activity compared to wildtype
MDA-9). In embodiments, the MDA-9 protein encoded by the SDCBP gene
has the amino acid sequence set forth in or corresponding to Entrez
6386, UniProt 000560, or RefSeq (protein) NP_001007068.1. In
embodiments, the SDCBP gene has the nucleic acid sequence set forth
in RefSeq (mRNA) NM_001007067.1. In embodiments, the amino acid
sequence or nucleic acid sequence is the sequence known at the time
of filing of the present application. In embodiments, the MDA-9
protein includes the sequence of SEQ ID NO: 1.
[0213] The term "PDZ domain" as referred to herein refers to a
common structural domain typically including 80-90 amino acids. PDZ
domains are common to scaffold and signaling proteins and play an
important role in signal transduction complexes. PDZ domains of a
protein can facilitate protein-protein interactions by binding
target proteins. MDA-9 proteins include two tandem PDZ domains,
PDZ1 and PDZ2, respectively, separated by a sequence of amino acids
linking the two domains. The amino acid sequence linking the PDZ1
and PDZ2 domains is referred to herein as an "interface region.".
In embodiments, the amino acid sequence of PDZ1 includes the
sequence of SEQ ID NO: 1. In embodiments, the amino acid sequence
of PDZ1 is the sequence of SEQ ID NO: 1.
[0214] "Biological sample" or "sample" refer to materials obtained
from or derived from a subject or patient. A biological sample
includes sections of tissues such as biopsy and autopsy samples,
and frozen sections taken for histological purposes. Such samples
include bodily fluids such as blood and blood fractions or products
(e.g., serum, plasma, platelets, red blood cells, and the like),
sputum, tissue, cultured cells (e.g., primary cultures, explants,
and transformed cells) stool, urine, synovial fluid, joint tissue,
synovial tissue, synoviocytes, fibroblast-like synoviocytes,
macrophage-like synoviocytes, immune cells, hematopoietic cells,
fibroblasts, macrophages, T cells, etc. A biological sample is
typically obtained from a eukaryotic organism, such as a mammal
such as a primate e.g., chimpanzee or human; cow; dog; cat; a
rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile;
or fish.
[0215] A "control" sample or value refers to a sample that serves
as a reference, usually a known reference, for comparison to a test
sample. For example, a test sample can be taken from a test
condition, e.g., in the presence of a test compound, and compared
to samples from known conditions, e.g., in the absence of the test
compound (negative control), or in the presence of a known compound
(positive control). A control can also represent an average value
gathered from a number of tests or results. One of skill in the art
will recognize that controls can be designed for assessment of any
number of parameters. For example, a control can be devised to
compare therapeutic benefit based on pharmacological data (e.g.,
half-life) or therapeutic measures (e.g., comparison of side
effects). One of skill in the art will understand which controls
are valuable in a given situation and be able to analyze data based
on comparisons to control values. Controls are also valuable for
determining the significance of data. For example, if values for a
given parameter are widely variant in controls, variation in test
samples will not be considered as significant.
[0216] "Patient" or "subject in need thereof" refers to a living
organism suffering from or prone to a disease or condition that can
be treated by administration of a compound, composition, or
pharmaceutical composition as provided herein. Non-limiting
examples include humans, other mammals, bovines, rats, mice, dogs,
monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
In some embodiments, a patient is human.
[0217] The terms "disease" or "condition" refer to a state of being
or health status of a patient or subject capable of being treated
with a compound, pharmaceutical composition, or method provided
herein. In embodiments, the disease is cancer (e.g. melanoma,
glioblastoma, head and neck cancer, urothelial cancer, breast
cancer, uveal melanoma, gastric cancer, lung adenocarcinoma,
hepatocellular carcinoma, colorectal cancer, prostate cancer,
pancreatic cancer, or neuroblastoma).
[0218] The terms "treating", or "treatment" refers to any indicia
of success in the treatment or amelioration of an injury, disease,
pathology or condition, including any objective or subjective
parameter such as abatement; remission; diminishing of symptoms or
making the injury, pathology or condition more tolerable to the
patient; slowing in the rate of degeneration or decline; making the
final point of degeneration less debilitating; improving a
patient's physical or mental well-being. The treatment or
amelioration of symptoms can be based on objective or subjective
parameters; including the results of a physical examination,
neuropsychiatric exams, and/or a psychiatric evaluation. The term
"treating" and conjugations thereof, include prevention of an
injury, pathology, condition, or disease. In embodiments,
"treating" refers to treatment of cancer. In embodiments,
"treating" refers to treatment of infectious disease. In
embodiments, "treating" refers to treatment of neurodegenerative
disease. In embodiments, "treating" refers to treatment of
inflammatory disease.
[0219] In embodiments, treatment or treating includes (1)
inhibiting a disease in a subject or patient experiencing or
displaying the pathology or symptomatology of cancer (e.g.,
arresting further development of the pathology and/or
symptomatology), (2) ameliorating a disease in a subject or patient
that is experiencing or displaying the pathology or symptomatology
of cancer (e.g., reversing the pathology and/or symptomatology),
and/or (3) effecting any measurable decrease in a disease in a
subject or patient that is experiencing or displaying the pathology
or symptomatology of cancer. In embodiments, the subject treated as
described herein may also fully recover from cancer and may become
cancer-free as a result of the present methods.
[0220] In embodiments, treating cancer refers to at least
ameliorating and/or decreasing and/or eradicating aspects of the
disease such as the following: the size of a tumor may be lessened
and/or the tumor may be completely destroyed; remnants of a tumor
(e.g. after surgery) may be lessened and/or destroyed; the growth
of a tumor may be prevented and/or the growth rate may be slowed;
the metastatic potential of a tumor may be decreased or eliminated;
cancer cells may be sensitized to radiation therapy, etc. For
example, when cancer cells are exposed to a compound or drug
described herein prior to, during or after radiation therapy, they
are more susceptible to killing by radiation, e.g. at least about
25% more of the cancer cells die without dividing, and typically at
least about 50, 75 or even 100% of the cells die without dividing,
compared to the number that die when exposed to radiation alone. In
addition, in embodiments, select changes which typically occur in
cancer cells when exposed to radiation are decreased or eliminated
when a compound or drug described herein is administered to a
subject receiving radiotherapy. For example, cancer cells
frequently exhibit an increased ability to grow, divide, and/or
metastasize after radiation therapy, and administration of the
present drugs (e.g., compound described herein) attenuates or
eliminates this ability. In embodiments, the treatment of cancer
metastasis includes the treatment of at least one of invasion,
migration, and angiogenesis.
[0221] A "effective amount" is an amount sufficient for a compound
to accomplish a stated purpose relative to the absence of the
compound (e.g. achieve the effect for which it is administered,
treat a disease, reduce enzyme activity, increase enzyme activity,
reduce a signaling pathway, or reduce one or more symptoms of a
disease or condition). An example of an "effective amount" is an
amount sufficient to contribute to the treatment, prevention, or
reduction of a symptom or symptoms of a disease, which could also
be referred to as a "therapeutically effective amount." For
example, for a given parameter, a therapeutically effective amount
will show an increase or decrease of at least 5%, 10%, 15%, 20%,
25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic
efficacy can also be expressed as "-fold" increase or decrease. For
example, a therapeutically effective amount can have at least a
1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
A "reduction" of a symptom or symptoms (and grammatical equivalents
of this phrase) means decreasing of the severity or frequency of
the symptom(s), or elimination of the symptom(s). A
"prophylactically effective amount" of a drug is an amount of a
drug that, when administered to a subject, will have the intended
prophylactic effect, e.g., preventing or delaying the onset (or
reoccurrence) of an injury, disease, pathology or condition, or
reducing the likelihood of the onset (or reoccurrence) of an
injury, disease, pathology, or condition, or their symptoms. The
full prophylactic effect does not necessarily occur by
administration of one dose, and may occur only after administration
of a series of doses. Thus, a prophylactically effective amount may
be administered in one or more administrations. An "activity
decreasing amount," as used herein, refers to an amount of
antagonist required to decrease the activity of an enzyme relative
to the absence of the antagonist. A "function disrupting amount,"
as used herein, refers to the amount of antagonist or inhibitor
(e.g., PDZ1 domain binder) required to disrupt the function of an
enzyme or protein (e.g., MDA-9) relative to the absence of the
antagonist. The exact amounts will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art
using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage
Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of
Pharmaceutical Compounding (1999); Pickar, Dosage Calculations
(1999); and Remington: The Science and Practice of Pharmacy, 20th
Edition, 2003, Gennaro, Ed., Lippincott, Williams &
Wilkins).
[0222] For any compound described herein, the therapeutically
effective amount can be initially determined from cell culture
assays. Target concentrations will be those concentrations of
active compound(s) that are capable of achieving the methods
described herein, as measured using the methods described herein or
known in the art.
[0223] As is well known in the art, therapeutically effective
amounts for use in humans can also be determined from animal
models. For example, a dose for humans can be formulated to achieve
a concentration that has been found to be effective in animals. The
dosage in humans can be adjusted by monitoring compounds
effectiveness and adjusting the dosage upwards or downwards, as
described above. Adjusting the dose to achieve maximal efficacy in
humans based on the methods described above and other methods is
well within the capabilities of the ordinarily skilled artisan.
[0224] Dosages may be varied depending upon the requirements of the
patient and the compound being employed. The dose administered to a
patient, in the context of the present disclosure, should be
sufficient to effect a beneficial therapeutic response in the
patient over time. The size of the dose also will be determined by
the existence, nature, and extent of any adverse side-effects.
Determination of the proper dosage for a particular situation is
within the skill of the practitioner. Generally, treatment is
initiated with smaller dosages which are less than the optimum dose
of the compound. Thereafter, the dosage is increased by small
increments until the optimum effect under circumstances is reached.
Dosage amounts and intervals can be adjusted individually to
provide levels of the administered compound effective for the
particular clinical indication being treated. This will provide a
therapeutic regimen that is commensurate with the severity of the
individual's disease state.
[0225] As used herein, the term "administering" means oral
administration, administration as a suppository, topical contact,
intravenous, parenteral, intraperitoneal, intramuscular,
intralesional, intrathecal, intranasal or subcutaneous
administration, or the implantation of a slow-release device, e.g.,
a mini-osmotic pump, to a subject. Administration is by any route,
including parenteral and transmucosal (e.g., buccal, sublingual,
palatal, gingival, nasal, vaginal, rectal, or transdermal).
Parenteral administration includes, e.g., intravenous,
intramuscular, intra-arteriole, intradermal, subcutaneous,
intraperitoneal, intraventricular, and intracranial. Other modes of
delivery include, but are not limited to, the use of liposomal
formulations, intravenous infusion, transdermal patches, etc. In
embodiments, the administering does not include administration of
any active agent other than the recited active agent.
[0226] "Co-administer" it is meant that a composition described
herein is administered at the same time, just prior to, or just
after the administration of one or more additional therapies. The
compounds provided herein can be administered alone or can be
coadministered to the patient. Coadministration is meant to include
simultaneous or sequential administration of the compounds
individually or in combination (more than one compound). Thus, the
preparations can also be combined, when desired, with other active
substances (e.g. to reduce metabolic degradation). The compositions
of the present disclosure can be delivered transdermally, by a
topical route, or formulated as applicator sticks, solutions,
suspensions, emulsions, gels, creams, ointments, pastes, jellies,
paints, powders, and aerosols.
[0227] As used herein, the term "cancer" refers to all types of
cancer, neoplasm or malignant tumors found in mammals, including
leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas
and sarcomas. Exemplary cancers that may be treated with a
compound, pharmaceutical composition, or method provided herein
include lymphoma, sarcoma, bladder cancer, bone cancer, brain
tumor, cervical cancer, colon cancer, esophageal cancer, gastric
cancer, head and neck cancer, kidney cancer, myeloma, thyroid
cancer, leukemia, prostate cancer, breast cancer (e.g. triple
negative, ER positive, ER negative, chemotherapy resistant,
herceptin resistant, HER2 positive, doxorubicin resistant,
tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary,
metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g.,
hepatocellular carcinoma), lung cancer (e.g. non-small cell lung
carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell
lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma),
glioblastoma multiforme, glioma, melanoma, prostate cancer,
castration-resistant prostate cancer, breast cancer, triple
negative breast cancer, glioblastoma, ovarian cancer, lung cancer,
squamous cell carcinoma (e.g., head, neck, or esophagus),
colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B
cell lymphoma, or multiple myeloma. Additional examples include,
cancer of the thyroid, endocrine system, brain, breast, cervix,
colon, head & neck, esophagus, liver, kidney, lung, non-small
cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus
or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,
multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme,
ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary
macroglobulinemia, primary brain tumors, cancer, malignant
pancreatic insulanoma, malignant carcinoid, urinary bladder cancer,
premalignant skin lesions, testicular cancer, lymphomas, thyroid
cancer, neuroblastoma, esophageal cancer, genitourinary tract
cancer, malignant hypercalcemia, endometrial cancer, adrenal
cortical cancer, neoplasms of the endocrine or exocrine pancreas,
medullary thyroid cancer, medullary thyroid carcinoma, melanoma,
colorectal cancer, papillary thyroid cancer, hepatocellular
carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular
Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate
cells, cancer of the hepatic stellate cells, or prostate
cancer.
[0228] The term "leukemia" refers broadly to progressive, malignant
diseases of the blood-forming organs and is generally characterized
by a distorted proliferation and development of leukocytes and
their precursors in the blood and bone marrow. Leukemia is
generally clinically classified on the basis of (1) the duration
and character of the disease-acute or chronic; (2) the type of cell
involved; myeloid (myelogenous), lymphoid (lymphogenous), or
monocytic; and (3) the increase or non-increase in the number
abnormal cells in the blood-leukemic or aleukemic (subleukemic).
Exemplary leukemias that may be treated with a compound,
pharmaceutical composition, or method provided herein include, for
example, acute nonlymphocytic leukemia, chronic lymphocytic
leukemia, acute granulocytic leukemia, chronic granulocytic
leukemia, acute promyelocytic leukemia, adult T-cell leukemia,
aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia,
blast cell leukemia, bovine leukemia, chronic myelocytic leukemia,
leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross'
leukemia, hairy-cell leukemia, hemoblastic leukemia,
hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia,
acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,
lymphoblastic leukemia, lymphocytic leukemia, lymphogenous
leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell
leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,
monocytic leukemia, myeloblastic leukemia, myelocytic leukemia,
myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli
leukemia, plasma cell leukemia, multiple myeloma, plasmacytic
leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's
leukemia, stem cell leukemia, subleukemic leukemia, or
undifferentiated cell leukemia.
[0229] The term "sarcoma" generally refers to a tumor which is made
up of a substance like the embryonic connective tissue and is
generally composed of closely packed cells embedded in a fibrillar
or homogeneous substance. Sarcomas that may be treated with a
compound, pharmaceutical composition, or method provided herein
include a chondrosarcoma, fibrosarcoma, lymphosarcoma,
melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma,
adipose sarcoma, liposarcoma, alveolar soft part sarcoma,
ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio
carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial
sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma,
fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma,
Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic
sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic
sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer
cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma
sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,
serocystic sarcoma, synovial sarcoma, or telangiectaltic
sarcoma.
[0230] The term "melanoma" is taken to mean a tumor arising from
the melanocytic system of the skin and other organs. Melanomas that
may be treated with a compound, pharmaceutical composition, or
method provided herein include, for example, acral-lentiginous
melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's
melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma,
lentigo maligna melanoma, malignant melanoma, nodular melanoma,
subungal melanoma, or superficial spreading melanoma.
[0231] The term "carcinoma" refers to a malignant new growth made
up of epithelial cells tending to infiltrate the surrounding
tissues and give rise to metastases. Exemplary carcinomas that may
be treated with a compound, pharmaceutical composition, or method
provided herein include, for example, medullary thyroid carcinoma,
familial medullary thyroid carcinoma, acinar carcinoma, acinous
carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,
carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar
carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma
basocellulare, basaloid carcinoma, basosquamous cell carcinoma,
bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic
carcinoma, cerebriform carcinoma, cholangiocellular carcinoma,
chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus
carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma
cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct
carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,
encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale
adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma
fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell
carcinoma, carcinoma gigantocellulare, glandular carcinoma,
granulosa cell carcinoma, hair-matrix carcinoma, hematoid
carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,
hyaline carcinoma, hypernephroid carcinoma, infantile embryonal
carcinoma, carcinoma in situ, intraepidermal carcinoma,
intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell
carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma
lenticulare, lipomatous carcinoma, lobular carcinoma,
lymphoepithelial carcinoma, carcinoma medullare, medullary
carcinoma, melanotic carcinoma, carcinoma molle, mucinous
carcinoma, carcinoma muciparum, carcinoma mucocellulare,
mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma,
carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell
carcinoma, carcinoma ossificans, osteoid carcinoma, papillary
carcinoma, periportal carcinoma, preinvasive carcinoma, prickle
cell carcinoma, pultaceous carcinoma, renal cell carcinoma of
kidney, reserve cell carcinoma, carcinoma sarcomatodes,
Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti,
signet-ring cell carcinoma, carcinoma simplex, small-cell
carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle
cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticum,
carcinoma telangiectodes, transitional cell carcinoma, carcinoma
tuberosum, tubular carcinoma, tuberous carcinoma, verrucous
carcinoma, or carcinoma villosum.
[0232] As used herein, the terms "metastasis," "metastatic," and
"metastatic cancer" can be used interchangeably and refer to the
spread of a proliferative disease or disorder, e.g., cancer, from
one organ or another non-adjacent organ or body part Cancer occurs
at an originating site, e.g., breast which site is referred to as a
primary tumor, e.g., primary breast cancer. Some cancer cells in
the primary tumor or originating site acquire the ability to
penetrate and infiltrate surrounding normal tissue in the local
area and/or the ability to penetrate the walls of the lymphatic
system or vascular system circulating through the system to other
sites and tissues in the body. A second clinically detectable tumor
formed from cancer cells of a primary tumor is referred to as a
metastatic or secondary tumor. When cancer cells metastasize, the
metastatic tumor and its cells are presumed to be similar to those
of the original tumor. Thus, if lung cancer metastasizes to the
breast, the secondary tumor at the site of the breast consists of
abnormal lung cells and not abnormal breast cells. The secondary
tumor in the breast is referred to a metastatic lung cancer. Thus,
the phrase metastatic cancer refers to a disease in which a subject
has or had a primary tumor and has one or more secondary tumors.
The phrases non-metastatic cancer or subjects with cancer that is
not metastatic refers to diseases in which subjects have a primary
tumor but not one or more secondary tumors. For example, metastatic
lung cancer refers to a disease in a subject with or with a history
of a primary lung tumor and with one or more secondary tumors at a
second location or multiple locations, e.g., in the breast.
[0233] In embodiments, a patient who is treated by the compounds
described herein, including embodiments thereof (e.g., as pure
drugs, salts, or prodrugs), and methods disclosed herein suffers
from cancer. Examples of cancer that may be so treated include but
are not limited to: adrenal cortical cancer, anal cancer, bile duct
cancer (e.g. peripheral cancer, distal bile duct cancer,
intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g.
osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma,
osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous
histiocytoma, giant cell tumor of the bone, chordoma, lymphoma,
multiple myeloma), brain and central nervous system cancer (e.g.
meningioma, astocytoma, oligodendrogliomas, ependymoma, glioma,
glioblastoma, medulloblastoma, ganglioglioma, Schwannoma,
germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma
in situ, infiltrating ductal carcinoma, infiltrating lobular
carcinoma, lobular carcinoma in situ, gynecomastia), Castleman
disease (e.g. giant lymph node hyperplasia, angiofollicular lymph
node hyperplasia), cervical cancer, colorectal cancer, endometrial
cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary
serous adnocarcinoma, clear cell), esophagus cancer, gallbladder
cancer (mucinous adenocarcinoma, small cell carcinoma),
gastrointestinal carcinoid tumors (e.g. choriocarcinoma,
chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's
lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer),
laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma,
hepatic adenoma, focal nodular hyperplasia, hepatocellular
carcinoma), lung cancer (e.g. small cell lung cancer, non-small
cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and
paranasal sinus cancer (e.g. esthesioneuroblastoma, midline
granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and
oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile
cancer, pituitary cancer, prostate cancer, retinoblastoma,
rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar
rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland
cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer),
stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ
cell cancer), thymus cancer, thyroid cancer (e.g. follicular
carcinoma, anaplastic carcinoma, poorly differentiated carcinoma,
medullary thyroid carcinoma, thyroid lymphoma), urothelial cell
cancer, vaginal cancer, vulvar cancer, and uterine cancer (e.g.
uterine leiomyosarcoma). Generally, the cancer is characterized by
the presence of at least one solid tumor.
[0234] The term "associated" or "associated with" in the context of
a substance or substance activity or function associated with a
disease (e.g., cancer (e.g. melanoma, glioblastoma, head and neck
cancer, urothelial cancer, breast cancer, uveal melanoma, gastric
cancer, lung adenocarcinoma, hepatocellular carcinoma, colorectal
cancer, prostate cancer, pancreatic cancer, or neuroblastoma.)
means that the disease (e.g. melanoma, glioblastoma, head and neck
cancer, urothelial cancer, breast cancer, uveal melanoma, gastric
cancer, lung adenocarcinoma, hepatocellular carcinoma, colorectal
cancer, prostate cancer, pancreatic cancer, or neuroblastoma.) is
caused by (in whole or in part), or a symptom of the disease is
caused by (in whole or in part) the substance or substance activity
or function.
[0235] "Anti-cancer agent" is used in accordance with its plain
ordinary meaning and refers to a composition (e.g. compound, drug,
antagonist, inhibitor, modulator) having antineoplastic properties
or the ability to inhibit the growth or proliferation of cells. In
embodiments, an anti-cancer agent is a chemotherapeutic. In
embodiments, an anti-cancer agent is an agent identified herein
having utility in methods of treating cancer. In embodiments, an
anti-cancer agent is an agent approved by the FDA or similar
regulatory agency of a country other than the USA, for treating
cancer.
[0236] The compositions described herein can be used in combination
with one another, with other active agents known to be useful in
treating a cancer such as anti-cancer agents.
[0237] Examples of anti-cancer agents include, but are not limited
to, radiation, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors
(e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244,
GSK1120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330,
PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY
869766), alkylating agents (e.g., cyclophosphamide, ifosfamide,
chlorambucil, busulfan, melphalan, mechlorethamine, uramustine,
thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine,
cyclophosphamide, chlorambucil, meiphalan), ethylenimine and
methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl
sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine,
lomusitne, semustine, streptozocin), triazenes (decarbazine)),
anti-metabolites (e.g., 5-azathioprine, leucovorin, capecitabine,
fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid
analog (e.g., methotrexate), or pyrimidine analogs (e.g.,
fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,
mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids
(e.g., vincristine, vinblastine, vinoielbine, vindesine,
podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase
inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide
(VP16), etoposide phosphate, teniposide, etc.), antitumor
antibiotics (e.g., doxorubicin, adriamycin, daunorubicin,
epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone,
plicamycin, etc.), platinum-based compounds (e.g. cisplatin,
oxaloplatin, carboplatin), anthracenedione (e.g., mitoxantrone),
substituted urea (e.g., hydroxyurea), methyl hydrazine derivative
(e.g., procarbazine), adrenocortical suppressant (e.g., mitotane,
aminoglutethimide), epipodophyllotoxins (e.g., etoposide),
antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes
(e.g., L-asparaginase), inhibitors of mitogen-activated protein
kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901,
ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or
LY294002, Syk inhibitors, mTOR inhibitors, antibodies (e.g.,
rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, all
trans-retinoic acid (ATRA), bryostatin, tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL),
5-aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin,
vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.),
geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG),
flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082,
PKC412, PD184352, 20-epi-1, 25 dihydroxyvitamin D3;
5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;
adozelesin; aldesleukin; ALL-TK antagonists; altretamine;
ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin;
amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis
inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing
morphogenetic protein-1; antiandrogen, prostatic carcinoma; anti
estrogen; antineoplaston; antisense oligonucleotides; aphidicolin
glycinate; apoptosis gene modulators; apoptosis regulators;
apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;
atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin
3; azasetron; azatoxin; azatyrosine; baccatin III derivatives;
balanol; batimastat; BCR/ABL antagonists; benzochlorins;
benzoylstaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide;
bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
bizelesin; breflate; bropirimine; budotitane; buthionine
sulfoximine; calcipotriol; calphostin C; camptothecin derivatives;
canarypox IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins;
chloroquinoxaline sulfonamide; cicapiost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine;
docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;
duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;
eflomithine; elemene; emitefur; epirubicin; epristeride;
estramustine analogue; estrogen agonists; estrogen antagonists;
etanidazole; etoposide phosphate; exemestane; fadrozole;
fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;
flezelastine; fluasterone; fludarabine; fluorodaunorunicin
hydrochloride; forfenimex; formestane; fostriecin; fotemustine;
gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;
gelatinase inhibitors; gemcitabine; glutathione inhibitors;
hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;
ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;
ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;
insulin-like growth factor-1 receptor inhibitor; interferon
agonists; interferons; interleukins; iobenguane; iododoxorubicin;
ipomeanol, 4-; iroplact; irsogladine; isobengazole;
isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;
lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting
factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; M1F inhibitor, mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphteipin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer, ormaplatin; osaterone;
oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platimun-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin 32;
proteasome inhibitors; protein A-based immune modulator, protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras famesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor,
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RH ietinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNLJ;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain
antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate;
sodium phenylacetate; solverol; somatomedin binding protein;
sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulflnosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafiir; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; zinostatin stimalamer,
Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,
acivicin; aclarubicin; acodazole hydrochloride; acronine;
adozelesin; aldesleukin; altretamine; ambomycin; ametantrone
acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;
dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin;
dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin;
doxorubicin hydrochloride; droloxifene; droloxifene citrate;
dromostanolone propionate; duazomycin; edatrexate; eflomithine
hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;
epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;
estramustine; estramustine phosphate sodium; etanidazole;
etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;
fazarabine; fenretinide; floxuridine; fludarabine phosphate;
fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;
gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin
hydrochloride; ifosfamide; iimofosine; interleukin II (including
recombinant interleukin II, or rlL.sub.2), interferon alfa-2a;
interferon alfa-2b; interferon alfa-n1; interferon alfa-n3;
interferon beta-la; interferon gamma-1b; iproplatin; irinotecan
hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;
liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazoie; nogalamycin;
ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine;
peplomycin sulfate; perfosfamide; pipobroman; piposulfan;
piroxantrone hydrochloride; plicamycin; plomestane; porfimer
sodium; porfiromycin; prednimustine; procarbazine hydrochloride;
puromycin; puromycin hydrochloride; pyrazofurin; riboprine;
rogletimide; safingol; safingol hydrochloride; semustine;
simtrazene; sparfosate sodium; sparsomycin; spirogermanium
hydrochloride; spiromustine; spiroplatin; streptonigrin;
streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur;
teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;
testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;
tirapazamine; toremifene citrate; trestolone acetate; triciribine
phosphate; trimetrexate; trimetrexate glucuronate; triptorelin;
tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;
verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;
vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;
vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;
vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin
hydrochloride, agents that arrest cells in the G2-M phases and/or
modulate the formation or stability of microtubules, (e.g.
Taxol.TM. (i.e. paclitaxel), Taxotere.TM., compounds comprising the
taxane skeleton, Erbulozole (i.e. R-55104), Dolastatin 10 (i.e.
DLS-10 and NSC-376128), Mivobulin isethionate (i.e. as CI-980),
Vincristine, NSC-639829, Discodermolide (i.e. as NVP-XX-A-2%),
ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and
Altorhyrtin C), Spongistatins (e.g. Spongistatin 1, Spongistatin 2,
Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6,
Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin
hydrochloride (i.e. LU-103793 and NSC-D-669356), Epothilones (e.g.
Epothilone A, Epothilone B, Epothilone C (i.e. desoxyepothilone A
or dEpoA), Epothilone D (i.e. KOS-862, dEpoB, and desoxyepothilone
B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A
N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e.
BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F and
dEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663),
Soblidotin (i.e. TZT-1027), Cryptophycin 52 (i.e. LY-355703),
Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e.
NSC-106969), Oncocidin A1 (i.e. BTO-956 and DIME), Fijianolide B,
Laulimalide, Narcosine (also known as NSC-5366), Nascapine,
Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol,
lnanocine (i.e. NSC-698666), Eleutherobins (such as
Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and
Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B,
Diazonamide A, Taccalonolide A, Diozostatin, (-)-Phenylahistin
(i.e. NSCL-96F037), Myoseverin B, Resverastatin phosphate sodium,
steroids (e.g., dexamethasone), finasteride, aromatase inhibitors,
gonadotropin-releasing hormone agonists (GnRH) such as goserelin or
leuprolide, adrenocorticosteroids (e.g., prednisone), progestins
(e.g., hydroxyprogesterone caproate, megestrol acetate,
medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol,
ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens
(e.g., testosterone propionate, fluoxymesterone), antiandrogen
(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin
(BCG), levamisole, interleukin-2, alpha-interferon, etc.),
monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52,
anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins
(e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate,
anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate,
etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody
conjugated to .sup.111In, .sup.90Y, or .sup.131I, etc.),
triptolide, homoharringtonine, dactinomycin, doxorubicin,
epirubicin, topotecan, itraconazole, vindesine, cerivastatin,
vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,
clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib,
erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor
receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib
(Iressa.TM.), erlotinib (Tarceva.TM.), cetuximab (Erbitux.TM.),
lapatinib (Tykerb.TM.), panitumumab (Vectibix.TM.), vandetanib
(Caprelsa.TM.), afatinib/BIBW2992, CI-1033/canertinib,
neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543,
ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl
erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040,
WZ4002, WZ3146, AG-490, XL647, PD1S3033, BMS-599626), S-FU, MCL-1
inhibitor, sorafenib, imatinib, sunitinib, dasatinib, or the like.
In embodiments, the compositions herein may be used in combination
with adjunctive agents that may not be effective alone, but may
contribute to the efficacy of the active agent in treating
cancer.
[0238] "Chemotherapeutic" or "chemotherapeutic agent" is used in
accordance with its plain ordinary meaning and refers to a chemical
composition or compound having antineoplastic properties or the
ability to inhibit the growth or proliferation of cells.
[0239] As used herein, the term "inflammatory disease" refers to a
disease or condition characterized by aberrant inflammation (e.g.
an increased level of inflammation compared to a control such as a
healthy person not suffering from a disease). Examples of
inflammatory diseases include traumatic brain injury, arthritis,
rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic
arthritis, multiple sclerosis, systemic lupus erythematosus (SLE),
myasthenia gravis, juvenile onset diabetes, diabetes mellitus type
1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's
thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,
vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet's
disease, Crohn's disease, ulcerative colitis, bullous pemphigoid,
sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel
disease, Addison's disease, Vitiligo, asthma, asthma, allergic
asthma, acne vulgaris, celiac disease, chronic prostatitis,
inflammatory bowel disease, pelvic inflammatory disease,
reperfusion injury, sarcoidosis, transplant rejection, interstitial
cystitis, atherosclerosis, and atopic dermatitis. In embodiments,
the inflammatory disease is asthma. In embodiments, the disease
associated with elevated expression of mda-9 is an inflammatory
disease.
[0240] As used herein, the term "neurodegenerative disorder" or
"neurodegenerative disease" refers to a disease or condition in
which the function of a subject's nervous system becomes impaired.
Examples of neurodegenerative diseases that may be treated with a
compound, pharmaceutical composition, or method described herein
include Alexander's disease, Alper's disease, Alzheimer's disease,
Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten
disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease),
Bovine spongiform encephalopathy (BSE), Canavan disease, chronic
fatigue syndrome, Cockayne syndrome, Corticobasal degeneration,
Creutzfeldt-Jakob disease, frontotemporal dementia,
Gerstmarm-Straussler-Scheinker syndrome, Huntington's disease,
HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru,
Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia
type 3), Multiple sclerosis, Multiple System Atrophy, myalgic
encephalomyelitis, Narcolepsy, Neuroborreliosis, Parkinson's
disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary
lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff's
disease, Schilder's disease, Subacute combined degeneration of
spinal cord secondary to Pernicious Anaemia, Schizophrenia,
Spinocerebellar ataxia (multiple types with varying
characteristics), Spinal muscular atrophy,
Steele-Richardson-Olszewski disease, progressive supranuclear
palsy, or Tabes dorsalis.
[0241] The term "infection" or "infectious disease" refers to a
disease or condition that can be caused by organisms such as a
bacterium, virus, fungi or any other pathogenic microbial agents.
In embodiments, the infectious diseases is a viral infection (e.g.,
HIV, SARS, HPV, influenza), or bacterial colonization in the human
gastrointestinal tract (e.g., pathenogenic bacterial colonization).
In embodiments, the infectious disease is associated with elevated
expression of mda-9. In embodiments, the infectious disease is
characterized by the presence of virus shedding (e.g., HIV viral
shedding or Herpes viral shedding). In embodiments, the infectious
disease is a bacterial infection. In embodiments, the infectious
disease is a gram-positive bacterial infection. In embodiments, the
infectious disease is a Staphylococcus aureus infection. In
embodiments, the infectious disease is Gram-positive or
Gram-negative bacterial infection. In embodiments, the infectious
disease is an infection associated with S. aureus, E. facium, E.
faecalis, K. pneumonoiaea, H. influenzaea, or P. aeruginosa. In
embodiments, the infectious disease is a S. aureus, E. facium, E.
faecalis, K. pneumonoiaea, H. influenzaea, or P. aeruginosa
infection.
II. Compounds
[0242] In an aspect is provided a compound, or pharmaceutically
acceptable salt thereof, having the formula:
##STR00005##
or a tautomer thereof.
[0243] Ring B is a cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl.
[0244] R.sup.1 is independently halogen, --CX.sup.1.sub.3,
--CHX.sup.1.sub.2, --CH.sub.2X.sup.1, --OCX.sup.1.sub.3,
--OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN, --SO.sub.n1R.sup.1D,
--SO.sub.v1NR.sup.1AR.sup.1B, --NHC(O)NR.sup.1AR.sup.1B,
--N(O).sub.m1, --NR.sup.1AR.sup.1B, --C(O)R.sup.1C,
--C(O)--OR.sup.1C, --C(O)NR.sup.1AR.sup.1B, --OR.sup.1D,
--NR.sup.1ASO.sub.2R.sup.1D, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)OR.sup.1C, --NR.sup.1AOR.sup.1C, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl. Two adjacent
R.sup.1 substituents may optionally be joined to form a substituted
or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl.
[0245] L.sup.1 is a bond, --S(O).sub.2--, --N(R.sup.3)--, --O--,
--S--, --C(O)--, --C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--,
--N(R.sup.3)C(O)NH--, --NHC(O)N(R.sup.3)--,
--S(O).sub.2N(R.sup.3)--, --N(R.sup.3)S(O).sub.2--,
--(O)S(O).sub.2N(R.sup.3)--, --N(R.sup.3)S(O).sub.2C(O)--,
--C(O)O--, --OC(O)--, substituted or unsubstituted alkylene,
substituted or unsubstituted heteroalkylene, substituted or
unsubstituted cycloalkylene, substituted or unsubstituted
heterocycloalkylene, substituted or unsubstituted arylene, or
substituted or unsubstituted heteroarylene.
[0246] R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --C(O)R.sup.3C,
--C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[0247] R.sup.5 is independently hydrogen, halogen,
--CX.sup.5.sub.3, --CHX.sup.5.sub.2, --CH.sub.2X.sup.5,
--OCX.sup.5.sub.3, --OCH.sub.2X.sup.5, --OCHX.sup.5.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl.
[0248] R.sup.6 is independently hydrogen,
halogen, --CX.sup.6.sub.3, --CHX.sup.6.sub.2, --CH.sub.2X.sup.6,
--OCX.sup.6.sub.3, --OCH.sub.2X.sup.6, --OCHX.sup.6.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl.
[0249] R.sup.5 and R.sup.6 substituents may optionally be joined to
form a substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[0250] R.sup.1A, R.sup.1B, R.sup.1C, R.sup.1D, R.sup.3A, R.sup.3B,
and R.sup.3C are independently hydrogen, --CX.sub.3, --CN, --COOH,
--CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl. R.sup.1A and R.sup.1B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl. R.sup.3A and R.sup.3B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl.
[0251] X, X.sup.1, X.sup.3, X.sup.5, and X.sup.6 are independently
--F, --Cl, --Br, or --I. The symbol n1 is an integer from 0 to 4.
The symbols m1 and v1 are independently 1 or 2. The symbol z1 is an
integer from 0 to 5.
[0252] In embodiments, Ring B is a (C.sub.3-C.sub.10) cycloalkyl, a
3 to 10 membered heterocycloalkyl, a (C.sub.6-C.sub.10) aryl, or a
5 to 10 membered heteroaryl. In embodiments, Ring B is a cycloalkyl
(e.g., C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl). In embodiments, Ring B is a
C.sub.3-C.sub.8 cycloalkyl. In embodiments, Ring B is a
C.sub.3-C.sub.6 cycloalkyl. In embodiments, Ring B is a
C.sub.5-C.sub.6 cycloalkyl. In embodiments, Ring B is a C.sub.6
cycloalkyl. In embodiments, Ring B is a C.sub.5 cycloalkyl. In
embodiments, Ring B is a (C.sub.6-C.sub.10) aryl. In embodiments,
Ring B is phenyl. In embodiments, Ring B is naphthyl. In
embodiments, Ring B is aziridinyl, oxiranyl, thiiranyl, azetidinyl,
oxetanyl, thietanyl, pyrrolidinyl, pyrrolyl, imidazolyl,
imidazolinyl, pyrazolinyl, tetrahydrofuranyl, thiolanyl,
piperidinyl, piperazinyl, pyranyl, morpholinyl, 1,4-dioxanyl,
tetrahydro-2H-pyranyl, thianyl, or dithianyl. In embodiments, Ring
B is a phenyl, thiofuranyl, imidazolyl, pyrazolyl, triazolyl,
tetrazolyl, furanyl, oxazolyl, isooxazolyl, oxadiazolyl,
oxatriazolyl, thienyl, thiazolyl, isothiazolyl, pyridinyl,
pyrazinyl, pyrimidinyl, pyridazinyl, or triazinyl (e.g.,
1,3,5-triazinyl, 1,2,3-triazinyl, or 1,2,4-triazinyl). In
embodiments, Ring B is indolyl, benzimidazolyl, indazolyl,
benzotriazolyl, pyrrolopyrimidinyl, purinyl, indolizinyl,
pyrrolopyriazinyl, pyrrolopyrimidinyl, imidazopyridazinyl,
imidazopyridinyl, imidazopyrimidinyl, cinnolinyl, quinazolinyl,
quinoxalinyl, phthalazinyl, pyridopyrazinyl, pteridinyl,
pyrazolopyridinyl, quinolinyl, isoquinolinyl, naphthyridinyl, or
carbazolyl. In embodiments, Ring B is
##STR00006##
[0253] In embodiments, R.sup.5 is hydrogen, halogen, unsubstituted
methyl, unsubstituted ethyl, unsubstituted propyl (e.g.,
unsubstituted n-propyl or unsubstituted isopropyl). In embodiments,
R.sup.5 is a substituted or unsubstituted C.sub.1-C.sub.6 alkyl. In
embodiments, R.sup.5 is substituted (e.g., substituted with a
substituent group, a size-limited substituent group, or lower
substituent group) or unsubstituted alkyl. In embodiments, R.sup.5
is substituted (e.g., substituted with a substituent group, a
size-limited substituent group, or lower substituent group) alkyl.
In embodiments, R.sup.5 is unsubstituted alkyl. In embodiments,
R.sup.5 is substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.5 is substituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.5 is unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2).
[0254] In embodiments, R.sup.5 is independently unsubstituted
methyl, unsubstituted ethyl, unsubstituted isopropyl, or
unsubstituted tert-butyl. In embodiments, R.sup.5 is independently
unsubstituted methyl. In embodiments, R.sup.5 is independently
unsubstituted ethyl. In embodiments, R.sup.5 is independently
unsubstituted propyl. In embodiments, R.sup.5 is independently
unsubstituted n-propyl. In embodiments, R.sup.5 is independently
unsubstituted isopropyl. In embodiments, R.sup.5 is independently
unsubstituted butyl. In embodiments, R.sup.5 is independently
unsubstituted n-butyl. In embodiments, R.sup.5 is independently
unsubstituted isobutyl. In embodiments, R.sup.5 is independently
unsubstituted tert-butyl. In embodiments, R.sup.5 is independently
unsubstituted pentyl. In embodiments, R.sup.5 is independently
unsubstituted hexyl. In embodiments, R.sup.5 is independently
unsubstituted heptyl. In embodiments, R.sup.5 is independently
unsubstituted octyl.
[0255] In embodiments, R.sup.5 is substituted (e.g., substituted
with a substituent group, a size-limited substituent group, or
lower substituent group) or unsubstituted heteroalkyl. In
embodiments, R.sup.5 is substituted (e.g., substituted with a
substituent group, a size-limited substituent group, or lower
substituent group) heteroalkyl. In embodiments, R.sup.5 is
unsubstituted heteroalkyl. In embodiments, R.sup.5 is substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered). In
embodiments, R.sup.5 is substituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.5 is an unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered).
[0256] In embodiments, R.sup.6 is hydrogen, halogen, unsubstituted
methyl, unsubstituted ethyl, unsubstituted propyl (e.g.,
unsubstituted n-propyl or unsubstituted isopropyl). In embodiments,
R.sup.6 is a substituted or unsubstituted C.sub.1-C.sub.6 alkyl. In
embodiments, R.sup.6 is substituted (e.g., substituted with a
substituent group, a size-limited substituent group, or lower
substituent group) or unsubstituted alkyl. In embodiments, R.sup.6
is substituted (e.g., substituted with a substituent group, a
size-limited substituent group, or lower substituent group) alkyl.
In embodiments, R.sup.6 is unsubstituted alkyl. In embodiments,
R.sup.6 is substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.6 is substituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.6 is unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2).
[0257] In embodiments, R.sup.6 is substituted (e.g., substituted
with a substituent group, a size-limited substituent group, or
lower substituent group) or unsubstituted heteroalkyl. In
embodiments, R.sup.6 is substituted (e.g., substituted with a
substituent group, a size-limited substituent group, or lower
substituent group) heteroalkyl. In embodiments, R.sup.6 is
unsubstituted heteroalkyl. In embodiments, R.sup.6 is substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered). In
embodiments, R.sup.6 is substituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.6 is an unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered).
[0258] In embodiments, R.sup.6 is independently unsubstituted
methyl, unsubstituted ethyl, unsubstituted isopropyl, or
unsubstituted tert-butyl. In embodiments, R.sup.6 is independently
unsubstituted methyl. In embodiments, R.sup.6 is independently
unsubstituted ethyl. In embodiments, R.sup.6 is independently
unsubstituted propyl. In embodiments, R.sup.6 is independently
unsubstituted n-propyl. In embodiments, R.sup.6 is independently
unsubstituted isopropyl. In embodiments, R.sup.6 is independently
unsubstituted butyl. In embodiments, R.sup.6 is independently
unsubstituted n-butyl. In embodiments, R.sup.6 is independently
unsubstituted isobutyl. In embodiments, R.sup.6 is independently
unsubstituted tert-butyl. In embodiments, R.sup.6 is independently
unsubstituted pentyl. In embodiments, R.sup.6 is independently
unsubstituted hexyl. In embodiments, R.sup.6 is independently
unsubstituted heptyl. In embodiments, R.sup.6 is independently
unsubstituted octyl.
[0259] In embodiments, R.sup.5 and R.sup.6 are joined to form a
substituted or unsubstituted cyclopropyl, substituted or
unsubstituted cyclopentyl, or substituted or unsubstituted
cyclohexyl. In embodiments, R.sup.5 and R.sup.6 are joined to form
a substituted cyclopropyl, substituted cyclobutyl, substituted
cyclopentyl, or substituted cyclohexyl. In embodiments, R.sup.5 and
R.sup.6 are joined to form an unsubstituted cyclopropyl,
unsubstituted cyclobutyl, an unsubstituted cyclopentyl, or an
unsubstituted cyclohexyl. In embodiments, R.sup.5 and R.sup.6 are
joined to form a substituted cyclopropyl, substituted cyclopentyl,
or substituted cyclohexyl. In embodiments, R.sup.5 and R.sup.6 are
joined to form an unsubstituted cyclopropyl, an unsubstituted
cyclopentyl, or an unsubstituted cyclohexyl.
[0260] In embodiments, R.sup.5 and R.sup.6 are joined to form a
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.4, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.5 and R.sup.6 are joined to form a substituted
(e.g., substituted with a substituent group, a size-limited
substituent group, or lower substituent group) or unsubstituted
cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.5 and
R.sup.6 are joined to form a substituted (e.g., substituted with a
substituent group, a size-limited substituent group, or lower
substituent group) cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.5 and R.sup.6 are joined to form an
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6).
[0261] In embodiments, R.sup.5 and R.sup.6 are joined to form a
substituted (e.g., substituted with a substituent group, a
size-limited substituent group, or lower substituent group) or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.5 and R.sup.6 are joined to form a substituted
(e.g., substituted with a substituent group, a size-limited
substituent group, or lower substituent group) heterocycloalkyl
(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5
membered, or 5 to 6 membered). In embodiments, R.sup.5 and R.sup.6
are joined to form an unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered).
[0262] In embodiments, two adjacent R.sup.1 substituents are joined
to form a substituted or unsubstituted (C.sub.3-C.sub.10)
cycloalkyl, substituted or unsubstituted 3 to 10 membered
heterocycloalkyl, substituted or unsubstituted (C.sub.6-C.sub.10)
aryl, or substituted or unsubstituted 5 to 10 membered
heteroaryl.
[0263] In embodiments, two adjacent R.sup.1 substituents are joined
to form a substituted or unsubstituted aziridinyl, substituted or
unsubstituted oxitanyl, substituted or unsubstituted thiiranyl,
substituted or unsubstituted azetidinyl, substituted or
unsubstituted oxetanyl, substituted or unsubstituted thietanyl,
substituted or unsubstituted pyrrolidinyl, substituted or
unsubstituted pyrrolyl, substituted or unsubstituted imidazolyl,
substituted or unsubstituted imidazolinyl, substituted or
unsubstituted pyrazolinyl, substituted or unsubstituted
tetrahydrofuranyl, substituted or unsubstituted thiolanyl,
substituted or unsubstituted piperidinyl, substituted or
unsubstituted piperazinyl, substituted or unsubstituted pyranyl,
substituted or unsubstituted morpholinyl, substituted or
unsubstituted 1,4-dioxanyl, substituted or unsubstituted
tetrahydro-2H-pyranyl, substituted or unsubstituted thianyl, or
substituted or unsubstituted dithianyl. In embodiments, two
adjacent R.sup.1 substituents are joined to form a substituted
aziridinyl, substituted oxiranyl, substituted thiiranyl,
substituted azetidinyl, substituted oxetanyl, substituted
thietanyl, substituted pyrrolidinyl, substituted pyrrolyl,
substituted imidazolyl, substituted imidazolinyl, substituted
pyrazolinyl, substituted tetrahydrofuranyl, substituted thiolanyl,
substituted piperidinyl, substituted piperazinyl, substituted
pyranyl, substituted morpholinyl, substituted 1,4-dioxanyl,
substituted tetrahydro-2H-pyranyl, substituted thianyl, or
substituted dithianyl. In embodiments, two adjacent R.sup.1
substituents are joined to form an unsubstituted aziridinyl,
unsubstituted oxiranyl, unsubstituted thiiranyl, unsubstituted
azetidinyl, unsubstituted oxetanyl, unsubstituted thietanyl,
unsubstituted pyrrolidinyl, unsubstituted pyrrolyl, unsubstituted
imidazolyl, unsubstituted imidazolinyl, unsubstituted pyrazolinyl,
unsubstituted tetrahydrofuranyl, unsubstituted thiolanyl,
unsubstituted piperidinyl, unsubstituted piperazinyl, unsubstituted
pyranyl, unsubstituted morpholinyl, unsubstituted 1,4-dioxanyl,
unsubstituted tetrahydro-2H-pyranyl, unsubstituted thianyl, or
unsubstituted dithianyl.
[0264] In an aspect is provided a compound, or pharmaceutically
acceptable salt thereof, having the formula:
##STR00007##
[0265] R.sup.1 is independently halogen, --CX.sup.1.sub.3,
--CHX.sup.1.sub.2, --CH.sub.2X.sup.1, --OCX.sup.1.sub.3,
--OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN, --SO.sub.n1R.sup.1D,
--SO.sub.v1NR.sup.1AR.sup.1B, --NHC(O)NR.sup.1AR.sup.1B,
--N(O).sub.m1, --NR.sup.1AR.sup.1B, --C(O)R.sup.1C,
--C(O)--OR.sup.1C, --C(O)NR.sup.1AR.sup.1B, --OR.sup.1D,
--NR.sup.1ASO.sub.2R.sup.1D, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)O R.sup.1C, --NR.sup.1AOR.sup.1C, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl; two adjacent
R.sup.1 substituents may optionally be joined to form a substituted
or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl.
[0266] R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--SO.sub.n2R.sup.2D, --SO.sub.v2NR.sup.2AR.sup.2B,
--NHC(O)NR.sup.2AR.sup.2B, --N(O).sub.m2, --NR.sup.2AR.sup.2B,
--C(O)R.sup.2C, --C(O)--OR.sup.2C, --C(O)NR.sup.2AR.sup.2B,
--OR.sup.2D, --NR.sup.2ASO.sub.2R.sup.2D, --NR.sup.2AC(O)R.sup.2D,
--NR.sup.2AC(O)O R.sup.2C, --NR.sup.2AOR.sup.2C, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl.
[0267] L.sup.1 is a
bond, --S(O).sub.2--, --N(R.sup.3)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--, --N(R.sup.3)C(O)NH--,
--NHC(O)N(R.sup.3)--, --S(O).sub.2N(R.sup.3)--,
N(R.sup.3)S(O).sub.2--, --C(O)S(O).sub.2N(R.sup.3)--,
--N(R.sup.3)S(O).sub.2C(O)--, --C(O)O--, --OC(O)--, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, or substituted or unsubstituted
heteroarylene.
[0268] R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --C(O)R.sup.3C,
--C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[0269] L.sup.2 is a
bond, --S(O).sub.2--, --N(R.sup.4)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--, --N(R.sup.4)C(O)NH--,
--NHC(O)N(R.sup.4)--, --S(O).sub.2N(R.sup.4)--,
N(R.sup.4)S(O).sub.2--, --C(O)S(O).sub.2N(R.sup.4)--,
--N(R.sup.4)S(O).sub.2C(O)--, --C(O)O--, --OC(O)--, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, or substituted or unsubstituted
heteroarylene.
[0270] R.sup.4 is independently hydrogen, --CX.sup.4.sub.3,
--CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN, --C(O)R.sup.4C,
--C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[0271] R.sup.1A, R.sup.1B, R.sup.1C, R.sup.1D, R.sup.2A, R.sup.2B,
R.sup.2D, R.sup.3A, R.sup.3B, R.sup.3C, R.sup.4A, R.sup.4B, and
R.sup.4C are independently hydrogen, --CX.sub.3, --CN, --COOH,
--CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl. R.sup.1A and R.sup.1B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl. R.sup.2A and R.sup.2B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl. R.sup.3A and R.sup.3B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl. R.sup.4A and R.sup.4B
substituents bonded to the same nitrogen atom may optionally be
joined to form a substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl.
[0272] X, X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently
--F, --Cl, --Br, or --I. The symbols n1 and n2 are independently an
integer from 0 to 4. The symbols m1, m2, v1, and v2 are
independently 1 or 2. The symbol z1 is an integer from 0 to 4.
[0273] In embodiments, R.sup.1 is independently halogen,
--CX.sup.1.sub.3, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
--OCX.sup.1.sub.3, --OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN,
--SO.sub.n1R.sup.1D, --SO.sub.v1NR.sup.1AR.sup.1B,
--NHC(O)NR.sup.1AR.sup.1B, --N(O).sub.m1, --NR.sup.1AR.sup.1B,
--C(O)R.sup.1C, --C(O)--OR.sup.1C, --C(O)NR.sup.1AR.sup.1B,
--OR.sup.1D, --NR.sup.1ASO.sub.2R.sup.id, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)O R.sup.1C, --NR.sup.1AOR.sup.1C, --N.sub.3,
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0274] In embodiments, R.sup.1 is independently halogen,
--CX.sup.1.sub.3, --CN, --OH, --NH.sub.2, --SH, --OCX.sup.1.sub.3,
--OCHX.sup.1.sub.2, --OCH.sub.2X.sup.1, --CHX.sup.1.sub.2,
--CH.sub.2.times. substituted or unsubstituted C.sub.1-C.sub.4
alkyl, substituted or unsubstituted 2 to 4 membered heteroalkyl,
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl,
substituted or unsubstituted 3 to 6 membered heterocycloalkyl,
substituted or unsubstituted phenyl, or substituted or
unsubstituted 5 to 6 membered heteroaryl. In embodiments, R.sup.1
is independently halogen, --CX.sup.1.sub.3, --CN, --OR.sup.2D,
--NH.sub.2, --SH, --OCX.sup.1.sub.3, --OCHX.sup.1.sub.2,
--OCH.sub.2X.sup.1, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
unsubstituted C.sub.1-C.sub.4 alkyl, or unsubstituted 2 to 4
membered heteroalkyl.
[0275] In embodiments, R.sup.1 is independently halogen,
--CX.sup.1.sub.3, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
--OCX.sup.1.sub.3, --OR.sup.2D, --CN, --NR.sup.1AR.sup.1B,
substituted or unsubstituted alkyl or two adjacent R.sup.1
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl. In embodiments, R.sup.1 is
independently halogen, --CX.sup.1.sub.3, --CHX.sup.1.sub.2,
--CH.sub.2X.sup.1, --OCX.sup.1.sub.3, --OR.sup.2D, --CN,
--NR.sup.1AR.sup.1B, or substituted or unsubstituted alkyl.
[0276] In embodiments, R.sup.1 is independently,
halogen, --CX.sup.1.sub.3, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
--OCX.sup.1.sub.3, --OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.20-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.20-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.20-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.20-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.20-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1 is independently hydrogen, halogen,
--CX.sup.1.sub.3, --CHX.sup.1.sub.2, --CH.sub.2X.sup.1,
--OCX.sup.1.sub.3, --OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.1 is independently --F, --Cl, --Br, or
--I.
[0277] In embodiments, R.sup.1 is independently hydrogen, halogen,
--NR.sup.1AR.sup.1B, --OR.sup.1D, or substituted or unsubstituted
heteroaryl. In embodiments, R.sup.1 is substituted or unsubstituted
aryl. In embodiments, R.sup.1 is substituted or unsubstituted
phenyl. In embodiments, R.sup.1 is hydrogen. In embodiments,
R.sup.1 is halogen. In embodiments, R.sup.1 is --NR.sup.1AR.sup.1B.
In embodiments, R.sup.1 is --OR.sup.1D. In embodiments, R.sup.1 is
substituted or unsubstituted heteroaryl. In embodiments, R.sup.1 is
substituted heteroaryl. In embodiments, R.sup.1 is substituted 5 to
6 membered heteroaryl. In embodiments, R.sup.1 is --NH.sub.2. In
embodiments, R.sup.1 is --OH. In embodiments, R.sup.1 is --Cl. In
embodiments, R.sup.1 is --F. In embodiments, R.sup.1 is
halogen.
[0278] In embodiments, R.sup.1 is R.sup.20-substituted or
unsubstituted alkyl or R.sup.20-substituted or unsubstituted
heteroalkyl. In embodiments, R.sup.1 is independently
R.sup.20-substituted methyl. In embodiments, R.sup.1 is
independently R.sup.20-substituted ethyl. In embodiments, R.sup.1
is independently unsubstituted methyl. In embodiments, R.sup.1 is
independently unsubstituted ethyl. In embodiments, R.sup.1 is
independently halogen, --OR.sup.2D, or --CH.sub.3. In embodiments,
R.sup.1 is halogen. In embodiments, R.sup.1 is --CH.sub.3. In
embodiments, R.sup.1 is --OR.sup.2D. In embodiments, R.sup.1 is
--OH.
[0279] In embodiments, R.sup.1 is R.sup.20-substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6
alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments, R.sup.1 is
R.sup.20-substituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R.sup.1 is an unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R.sup.1 is R.sup.20-substituted or unsubstituted heteroalkyl (e.g.,
2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4
membered heteroalkyl). In embodiments, R.sup.1 is
R.sup.20-substituted heteroalkyl (e.g., 2 to 8 membered
heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered
heteroalkyl). In embodiments, R.sup.1 is an unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments,
R.sup.1 is R.sup.20-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl). In embodiments, R.sup.1 is
R.sup.20-substituted cycloalkyl (e.g., C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6 cycloalkyl). In
embodiments, R.sup.1 is an unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl). In embodiments, R.sup.1 is
R.sup.20-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5
to 6 membered heterocycloalkyl). In embodiments, R.sup.1 is
R.sup.20-substituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl). In embodiments, R.sup.1 is an
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl). In embodiments, R.sup.1 is
R.sup.20-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
aryl, C.sub.10 aryl, or phenyl). In embodiments, R.sup.1 is
R.sup.20-substituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10
aryl, or phenyl). In embodiments, R.sup.1 is an unsubstituted aryl
(e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In
embodiments, R.sup.1 is R.sup.20-substituted or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R.sup.1
is R.sup.20-substituted heteroaryl (e.g., 5 to 10 membered
heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R.sup.1 is an unsubstituted heteroaryl
(e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or
5 to 6 membered heteroaryl).
[0280] In embodiments, R.sup.1 is R.sup.20-substituted or
unsubstituted methyl. In embodiments, R.sup.1 is
R.sup.20-substituted or unsubstituted C.sub.2 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted or unsubstituted
C.sub.3 alkyl. In embodiments, R.sup.1 is R.sup.20-substituted or
unsubstituted C.sub.4 alkyl. In embodiments, R.sup.1 is
R.sup.20-substituted or unsubstituted C.sub.5 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted or unsubstituted
C.sub.6 alkyl. In embodiments, R.sup.1 is R.sup.20-substituted or
unsubstituted C.sub.7 alkyl. In embodiments, R.sup.1 is
R.sup.20-substituted or unsubstituted C.sub.8 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted methyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.2 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.3 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.4 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.5 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.6 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.7 alkyl. In
embodiments, R.sup.1 is R.sup.20-substituted C.sub.8 alkyl. In
embodiments, R.sup.1 is an unsubstituted methyl. In embodiments,
R.sup.1 is an unsubstituted C.sub.2 alkyl. In embodiments, R.sup.1
is an unsubstituted C.sub.3 alkyl. In embodiments, R.sup.1 is an
unsubstituted C.sub.4 alkyl. In embodiments, R.sup.1 is an
unsubstituted C.sub.5 alkyl. In embodiments, R.sup.1 is an
unsubstituted Q alkyl. In embodiments, R.sup.1 is an unsubstituted
C.sub.7 alkyl. In embodiments, R.sup.1 is an unsubstituted C.sub.8
alkyl.
[0281] R.sup.20 is independently oxo, halogen, --CX.sup.20.sub.3,
--CHX.sup.20.sub.2, --CH.sub.2X.sup.20, --OCX.sup.20.sub.3,
--OCH.sub.2X.sup.20, --OCHX.sup.20.sub.2, --CN, --OH, --NH.sub.2,
--CO OH, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SC.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.21-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.21-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.21-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.21-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.21-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.21-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.20 is independently oxo, halogen,
--CX.sup.20.sub.3, --CHX.sup.20.sub.2, --CH.sub.2X.sup.20,
--OCX.sup.20.sub.3, --OCH.sub.2X.sup.20, --OCHX.sup.20.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.20 is independently --F, --Cl, --Br, or
--I.
[0282] In embodiments, R.sup.20 is R.sup.21-substituted or
unsubstituted alkyl or R.sup.21-substituted or unsubstituted
heteroalkyl. In embodiments, R.sup.20 is independently
R.sup.21-substituted methyl. In embodiments, R.sup.20 is
R.sup.21-substituted ethyl. In embodiments, R.sup.20 is
independently unsubstituted methyl. In embodiments, R.sup.20 is
independently unsubstituted ethyl.
[0283] R.sup.21 is independently oxo,
halogen, --CX.sup.21.sub.3, --CHX.sup.21.sub.2, --CH.sub.2X.sup.21,
--OCX.sup.21.sub.3, --OCH.sub.2X.sup.21, --OCHX.sup.21.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.22-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.22-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.22-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.22-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.22-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.21 is independently oxo, halogen,
--CX.sup.21.sub.3, --CHX.sup.21.sub.2, --CH.sub.2X.sup.21,
--OCX.sup.21.sub.3, --OCH.sub.2X.sup.21, --OCHX.sup.21.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.21 is independently --F,
--Cl, --Br, or --I.
[0284] In embodiments, R.sup.21 is R.sup.20-substituted or
unsubstituted alkyl or R.sup.20-substituted or unsubstituted
heteroalkyl. In embodiments, R.sup.21 is independently
R.sup.20-substituted methyl. In embodiments, R.sup.21 is
R.sup.22-substituted ethyl. In embodiments, R.sup.21 is
independently unsubstituted methyl. In embodiments, R.sup.21 is
independently unsubstituted ethyl.
[0285] R.sup.22 is independently oxo, halogen, --CX.sup.22.sub.3,
--CHX.sup.22.sub.2, --CH.sub.2X.sup.22, --OCX.sup.22.sub.3,
--OCH.sub.2X.sup.22, --OCHX.sup.22.sub.2, --CN, --OH, --NH.sub.2,
--COO H, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SC.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.22 is independently --F, --Cl, --Br, or
--I.
[0286] In embodiments, R.sup.1A is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.1A is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.1A is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.1A is independently
unsubstituted methyl. In embodiments, R.sup.1A is independently
unsubstituted ethyl. In embodiments, R.sup.1A is independently
unsubstituted propyl. In embodiments, R.sup.1A is independently
unsubstituted isopropyl. In embodiments, R.sup.1A is independently
unsubstituted tert-butyl. In embodiments, R.sup.1A is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.1A is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.1A is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.1A is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.1A is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.1A is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.1A is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.1A is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.1A is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.1A is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1A is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.1A is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1A is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1A is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1A is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0287] In embodiments, R.sup.1B is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.1B is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.1B is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.1B is independently
unsubstituted methyl. In embodiments, R.sup.1B is independently
unsubstituted ethyl. In embodiments, R.sup.1B is independently
unsubstituted propyl. In embodiments, R.sup.1B is independently
unsubstituted isopropyl. In embodiments, R.sup.1B is independently
unsubstituted tert-butyl. In embodiments, R.sup.1B is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.1B is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.1B is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.1B is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.1B is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.1B is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.1B is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.1B is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.1B is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.1B is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1B is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.1B is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1B is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1B is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1B is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0288] In embodiments, R.sup.1A and R.sup.1B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.1A and R.sup.1B substituents bonded to the same
nitrogen atom may be joined to form a substituted heterocycloalkyl
(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5
membered, or 5 to 6 membered). In embodiments, R.sup.1A and
R.sup.1B substituents bonded to the same nitrogen atom may be
joined to form an unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered).
[0289] In embodiments, R.sup.1A and R.sup.1B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.1A and R.sup.1B
substituents bonded to the same nitrogen atom may be joined to form
a substituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.1A and R.sup.1B
substituents bonded to the same nitrogen atom may be joined to form
an unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered).
[0290] In embodiments, R.sup.1C is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.1C is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.1C is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.1C is independently
unsubstituted methyl. In embodiments, R.sup.1C is independently
unsubstituted ethyl. In embodiments, R.sup.1C is independently
unsubstituted propyl. In embodiments, R.sup.1C is independently
unsubstituted isopropyl. In embodiments, R.sup.1C is independently
unsubstituted tert-butyl. In embodiments, R.sup.1C is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.1C is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.1C is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.1C is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.1C is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.1C is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.1C is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.1C is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.1C is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.1C is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1C is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.1C is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1C is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1C is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1C is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0291] In embodiments, R.sup.1D is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.1D is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.1D is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.1D is independently
unsubstituted methyl. In embodiments, R.sup.1D is independently
unsubstituted ethyl. In embodiments, R.sup.1D is independently
unsubstituted propyl. In embodiments, R.sup.1D is independently
unsubstituted isopropyl. In embodiments, R.sup.1D is independently
unsubstituted tert-butyl. In embodiments, R.sup.1D is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.1D is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.1D is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.1D is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.1D is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.1D is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.1D is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.1D is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.1D is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.1D is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1D is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.1D is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.1D is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1D is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.1D is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0292] In embodiments, R.sup.1A is independently hydrogen,
--CX.sup.1A.sub.3, --CHX.sup.1A.sub.2, --CH.sub.2X.sup.1A, --CN,
--COOH, --CONH.sub.2, R.sup.20A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.20A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.20A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6, R.sup.20A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.20A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.20A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1A is independently hydrogen, --CX.sup.1A.sub.3,
--CHX.sup.1A.sub.2, --CH.sub.2X.sup.1A, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.4,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.1A is independently --F,
--Cl, --Br, or --I.
[0293] In embodiments, R.sup.1A is independently hydrogen. In
embodiments, R.sup.1A is independently unsubstituted methyl. In
embodiments, R.sup.1A is independently unsubstituted ethyl.
[0294] In embodiments, R.sup.1A and R.sup.1B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.20A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.20-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1A and R.sup.1B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.1A and R.sup.1B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.20A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.1A and R.sup.1B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0295] R.sup.20A is independently oxo,
halogen, --CX.sup.20A.sub.3, --CHX.sup.20A.sub.2,
--CH.sub.2X.sup.20A, --OCX.sup.20A.sub.3, --OCH.sub.2X.sup.20A,
--OCHX.sup.20A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.21A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.21A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.21-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.21A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.21A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.21A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.20A is independently oxo, halogen,
--CX.sup.20A.sub.3, --CHX.sup.20A.sub.2, --CH.sub.2X.sup.20A,
--OCX.sup.20A.sub.3, --OCH.sub.2X.sup.20A, --OCHX.sup.20A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.20A is independently --F,
--Cl, --Br, or --I.
[0296] In embodiments, R.sup.20A is independently unsubstituted
methyl. In embodiments, R.sup.20A is independently unsubstituted
ethyl.
[0297] R.sup.21A is independently oxo, halogen, --CX.sup.21A.sub.3,
--CHX.sup.21A.sub.2, --CH.sub.2X.sup.21A, --OCX.sup.21A.sub.3,
--OCH.sub.2X.sup.21A, --OCHX.sup.21A.sub.2, --CN, --OH, --NH.sub.2,
--COOH, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.20-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.20-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.22A-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.22A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.22A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.22A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.21A is independently oxo, halogen,
--CX.sup.21A.sub.3, --CHX.sup.21A.sub.2, --CH.sub.2X.sup.21A,
--OCX.sup.21A.sub.3, --OCH.sub.2X.sup.21A, --OCHX.sup.21A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.21A is independently --F,
--Cl, --Br, or -I.
[0298] In embodiments, R.sup.21A is independently unsubstituted
methyl. In embodiments, R.sup.21A is independently unsubstituted
ethyl.
[0299] R.sup.22A is independently oxo, halogen, --CX.sup.22A.sub.3,
--CHX.sup.22A.sub.2, --CH.sub.2X.sup.22A, --OCX.sup.22A.sub.3,
--OCH.sub.2X.sup.22A, --OCHX.sup.22A.sub.2, --CN, --OH, --NH.sub.2,
--COOH, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.22A is independently --F, --Cl, --Br, or
--I.
[0300] In embodiments, R.sup.22A is independently unsubstituted
methyl. In embodiments, R.sup.22A is independently unsubstituted
ethyl.
[0301] In embodiments, R.sup.1B is independently hydrogen,
--CX.sup.1B.sub.3, --CHX.sup.1B.sub.2, --CH.sub.2X.sup.1B, --CN,
--COOH, --CONH.sub.2, R.sup.20B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.20B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.20B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.20B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.20B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1B is independently hydrogen, --CX.sup.1B.sub.3,
--CHX.sup.1B.sub.2, --CH.sub.2X.sup.1B, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.1B is independently --F,
--Cl, --Br, or --I.
[0302] In embodiments, R.sup.1B is independently hydrogen. In
embodiments, R.sup.1B is independently unsubstituted methyl. In
embodiments, R.sup.1B is independently unsubstituted ethyl.
[0303] In embodiments, R.sup.1A and R.sup.1B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.20B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.20B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1A and R.sup.1B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.1A and R.sup.1B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.20B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.1A and R.sup.1B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0304] R.sup.20B is independently oxo,
halogen, --CX.sup.20B.sub.3, --CHX.sup.20B.sub.2,
--CH.sub.2X.sup.20B, --OCX.sup.20B.sub.3, --OCH.sub.2X.sup.20B,
--OCHX.sup.20B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.21B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.21B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.21B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.21B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.21B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.21B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.20B is independently oxo, halogen,
--CX.sup.20B.sub.3, --CHX.sup.20B.sub.2, --CH.sub.2X.sup.20B,
--OCX.sup.20B.sub.3, --OCH.sub.2X.sup.20B, --OCHX.sup.20B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.3NH.sub.3, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.20B is independently --F,
--Cl, --Br, or --I.
[0305] In embodiments, R.sup.20B is independently unsubstituted
methyl. In embodiments, R.sup.20B is independently unsubstituted
ethyl.
[0306] R.sup.21B is independently oxo,
halogen, --CX.sup.21B.sub.3, --CHX.sup.21B.sub.2,
--CH.sub.2X.sup.21B, --OCX.sup.21B.sub.3, --OCH.sub.2X.sup.21B,
--OCHX.sup.21B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.20-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.22B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.22B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.22B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.22B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.22B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.21B is independently oxo, halogen,
--CX.sup.21B.sub.3, --CHX.sup.21B.sub.2, --CH.sub.2X.sup.21B,
--OCX.sup.21B.sub.3, --OCH.sub.2X.sup.21B, --OCHX.sup.21B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.21B is independently --F,
--Cl, --Br, or --I.
[0307] In embodiments, R.sup.21B is independently unsubstituted
methyl. In embodiments, R.sup.21B is independently unsubstituted
ethyl.
[0308] R.sup.22B is independently oxo,
halogen, --CX.sup.22B.sub.3, --CHX.sup.22B.sub.2,
--CH.sub.2X.sup.22B, --OCX.sup.22B.sub.3, --OCH.sub.2X.sup.22B,
--OCHX.sup.22B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.22B is independently --F, --Cl, --Br, or --I.
[0309] In embodiments, R.sup.22B is independently unsubstituted
methyl. In embodiments, R.sup.22B is independently unsubstituted
ethyl.
[0310] In embodiments, R.sup.1C is independently hydrogen,
--CX.sup.1C.sub.3, --CHX.sup.1C.sub.2, --CH.sub.2X.sup.1C, --CN,
--COOH, --CONH.sub.2, R.sup.20C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.20C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.20C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.20C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.20C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1C is independently hydrogen, --CX.sup.1C.sub.3,
--CHX.sup.1C.sub.2, --CH.sub.2X.sup.1C, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.1C is independently --F,
--Cl, --Br, or --I.
[0311] In embodiments, R.sup.1C is independently hydrogen. In
embodiments, R.sup.1C is independently unsubstituted methyl. In
embodiments, R.sup.1C is independently unsubstituted ethyl.
[0312] R.sup.20C is independently oxo,
halogen, --CX.sup.20C.sub.3, --CHX.sup.20C.sub.2,
--CH.sub.2X.sup.20C, --OCX.sup.20C.sub.3, --OCH.sub.2X.sup.20C,
--OCHX.sup.20C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.21C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.21C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.21C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.21C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.21C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.21C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.20C is independently oxo, halogen,
--CX.sup.20C.sub.3, --CHX.sup.20C.sub.2, --CH.sub.2X.sup.20C,
--OCX.sup.20C.sub.3, --OCH.sub.2X.sup.20C, --OCHX.sup.20C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.20C is independently --F,
--Cl, --Br, or --I.
[0313] In embodiments, R.sup.20C is independently unsubstituted
methyl. In embodiments, R.sup.20C is independently unsubstituted
ethyl.
[0314] R.sup.21C is independently oxo,
halogen, --CX.sup.21C.sub.3, --CHX.sup.21C.sub.2,
--CH.sub.2X.sup.21C, --OCX.sup.21C.sub.3, --OCH.sub.2X.sup.21C,
--OCHX.sup.21C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.3NH.sub.3,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.22C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.22C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.22C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.22C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.22C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.22C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.21C is independently oxo, halogen,
--CX.sup.21C.sub.3, --CHX.sup.21C.sub.2, --CH.sub.2X.sup.21C,
--OCX.sup.21C.sub.3, --OCH.sub.2X.sup.21C, --OCHX.sup.21C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.21C is independently --F,
--Cl, --Br, or --I.
[0315] In embodiments, R.sup.21C is independently unsubstituted
methyl. In embodiments, R.sup.21C is independently unsubstituted
ethyl.
[0316] R.sup.22C is independently oxo,
halogen, --CX.sup.22C.sub.2, --CHX.sup.22C.sub.2,
--CH.sub.2X.sup.22C, --OCX.sup.22C.sub.3, --OCH.sub.2X.sup.22C,
--OCHX.sup.22B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.22C is independently --F, --Cl, --Br, or --I.
[0317] In embodiments, R.sup.22C is independently unsubstituted
methyl. In embodiments, R.sup.22C is independently unsubstituted
ethyl.
[0318] In embodiments, R.sup.1D is independently hydrogen,
--CX.sup.1D.sub.3, --CHX.sup.1D.sub.2, --CH.sub.2X.sup.1D, --CN,
--COOH, --CONH.sub.2, R.sup.20D-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.20D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.20D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.20D-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.20D-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.1D is independently hydrogen, --CX.sup.1D.sub.3,
--CHX.sup.1D.sub.2, --CH.sub.2X.sup.1D, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.1D is independently --F,
--Cl, --Br, or --I.
[0319] In embodiments, R.sup.1D is independently hydrogen. In
embodiments, R.sup.1D is independently unsubstituted methyl. In
embodiments, R.sup.1D is independently unsubstituted ethyl.
[0320] R.sup.20C is independently oxo,
halogen, --CX.sup.20D.sub.3, --CHX.sup.20D.sub.2,
--CH.sub.2X.sup.200, --OCX.sup.20D.sub.3, --OCH.sub.2X.sup.20D,
--OCHX.sup.20D.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.21D-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.21D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.21D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.21D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.21D-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.21D-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.20D is independently oxo, halogen,
--CX.sup.20D.sub.3, --CHX.sup.20D.sub.2, --CH.sub.2X.sup.20D,
--OCX.sup.20D.sub.3, --OCH.sub.2X.sup.20D, --OCHX.sup.20D.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.20D is independently --F,
--Cl, --Br, or --I.
[0321] In embodiments, R.sup.20D is independently unsubstituted
methyl. In embodiments, R.sup.20D is independently unsubstituted
ethyl.
[0322] R.sup.21D is independently oxo,
halogen, --CX.sup.21D.sub.3, --CHX.sup.21D.sub.2,
--CH.sub.2X.sup.21D, --OCX.sup.21D.sub.3, --OCH.sub.2X.sup.21D,
--OCHX.sup.21D.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.22D-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.22D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.22D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.22D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.22D-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.22D-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.21D is independently oxo, halogen,
--CX.sup.21D.sub.3, --CHX.sup.21D.sub.2, --CH.sub.2X.sup.21D,
--OCX.sup.21D.sub.3, --OCH.sub.2X.sup.21D, --OCHX.sup.21D.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.21D is independently --F,
--Cl, --Br, or --I.
[0323] In embodiments, R.sup.21D is independently unsubstituted
methyl. In embodiments, R.sup.21D is independently unsubstituted
ethyl.
[0324] R.sup.22D is independently oxo,
halogen, --CX.sup.22D.sub.3, --CHX.sup.22D.sub.2,
--CH.sub.2X.sup.22D, --OCX.sup.22--OCH.sub.2X.sup.22D,
--OCHX.sup.22B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.22D is independently --F, --Cl, --Br, or --I.
[0325] In embodiments, R.sup.22D is independently unsubstituted
methyl. In embodiments, R.sup.22D is independently unsubstituted
ethyl.
[0326] In embodiments, R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--SO.sub.n2R.sup.2D, --SO.sub.v2NR.sup.2AR.sup.2B,
--NHC(O)NR.sup.2AR.sup.2B, --N(O).sub.m2, --NR.sup.2AR.sup.2B,
--C(O)R.sup.2C, --C(O)--OR.sup.2C, --C(O)NR.sup.2AR.sup.2B,
--OR.sup.2D, --NR.sup.2ASO.sub.2R.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O)O R.sup.2C, --NR.sup.2AOR.sup.2C, --N.sub.3,
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0327] In embodiments, R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CN, --OH, --NH.sub.2, --SH, --OCX.sup.2.sub.3,
--OCHX.sup.2.sub.2, --OCH.sub.2X.sup.2, --CHX.sup.2.sub.2,
--CH.sub.2X.sup.2, substituted or unsubstituted C.sub.1-C.sub.4
alkyl, or substituted or unsubstituted 2 to 4 membered heteroalkyl,
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl,
substituted or unsubstituted 3 to 6 membered heterocycloalkyl,
substituted or unsubstituted phenyl, or substituted or
unsubstituted 5 to 6 membered heteroaryl. In embodiments, R.sup.2
is independently hydrogen, halogen, --CX.sup.2.sub.3, --CN, --OH,
--NH.sub.2, --SH, --OCX.sup.2.sub.3, --OCHX.sup.2.sub.2,
--OCH.sub.2X.sup.2, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
unsubstituted C.sub.1-C.sub.4 alkyl, or unsubstituted 2 to 4
membered heteroalkyl.
[0328] In embodiments, R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2,
--NR.sup.2AR.sup.2B, --C(O)R.sup.2C, --C(O)--OR.sup.2C,
--C(O)NR.sup.2AR.sup.2B, --OR.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O)OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[0329] In embodiments, R.sup.2 is independently hydrogen, halogen,
--NR.sup.2AR.sup.2B, --C(O)R.sup.2C, --C(O)--OR.sup.2C,
--C(O)NR.sup.2AR.sup.2B, --OR.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O) OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[0330] In embodiments, R.sup.2 is independently hydrogen, halogen,
--NR.sup.2AR.sup.2B, --OR.sup.2D, or substituted or unsubstituted
heteroaryl. In embodiments, R.sup.2 is substituted or unsubstituted
aryl. In embodiments, R.sup.2 is substituted or unsubstituted
phenyl. In embodiments, R.sup.2 is hydrogen. In embodiments,
R.sup.2 is halogen. In embodiments, R.sup.2 is --NR.sup.2AR.sup.2B.
In embodiments, R.sup.2 is --OR.sup.2D. In embodiments, R.sup.2 is
substituted or unsubstituted heteroaryl. In embodiments, R.sup.2 is
substituted heteroaryl. In embodiments, R.sup.2 is substituted 5 to
6 membered heteroaryl. In embodiments, R.sup.2 is --NH.sub.2. In
embodiments, R.sup.2 is --OH. In embodiments, R.sup.2 is --Cl. In
embodiments, R.sup.2 is --F. In embodiments, R.sup.2 is
halogen.
[0331] In embodiments, R.sup.2 is substituted or unsubstituted
(C.sub.3-C.sub.10) cycloalkyl, substituted or unsubstituted 3 to 10
membered heterocycloalkyl, substituted or unsubstituted
(C.sub.6-C.sub.10) aryl, or substituted or unsubstituted 5 to 10
membered heteroaryl. In embodiments, R.sup.2 is a substituted or
unsubstituted heteroaryl. In embodiments, R.sup.2 is a substituted
or unsubstituted 5 to 6 membered heteroaryl. In embodiments,
R.sup.2 is a substituted or unsubstituted 5 membered
heteroaryl.
[0332] In embodiments, R.sup.2 is a substituted or unsubstituted
(C.sub.3-C.sub.10) cycloalkyl, a substituted or unsubstituted 3 to
10 membered heterocycloalkyl, a substituted or unsubstituted
(C.sub.6-C.sub.10) aryl, or a substituted or unsubstituted 5 to 10
membered heteroaryl. In embodiments, R.sup.2 is a substituted or
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6 cycloalkyl). In
embodiments, R.sup.2 is a substituted or unsubstituted
C.sub.3-C.sub.8 cycloalkyl. In embodiments, R.sup.2 is a
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl. In
embodiments, R.sup.2 is a substituted or unsubstituted
C.sub.5-C.sub.6 cycloalkyl. In embodiments, R.sup.2 is a
substituted or unsubstituted C.sub.6 cycloalkyl. In embodiments,
R.sup.2 is a substituted or unsubstituted C.sub.5 cycloalkyl. In
embodiments, R.sup.2 is a substituted or unsubstituted
(C.sub.6-C.sub.10) aryl. In embodiments, R.sup.2 is substituted or
unsubstituted phenyl. In embodiments, R.sup.2 is substituted or
unsubstituted naphthyl. In embodiments, R.sup.2 is substituted or
unsubstituted aziridinyl, substituted or unsubstituted oxiranyl,
substituted or unsubstituted thiiranyl, substituted or
unsubstituted azetidinyl, substituted or unsubstituted oxetanyl,
substituted or unsubstituted thietanyl, substituted or
unsubstituted pyrrolidinyl, substituted or unsubstituted pyrrolyl,
substituted or unsubstituted imidazolyl, substituted or
unsubstituted imidazolinyl, substituted or unsubstituted
pyrazolinyl, substituted or unsubstituted tetrahydrofuranyl,
substituted or unsubstituted thiolanyl, substituted or
unsubstituted piperidinyl, substituted or unsubstituted
piperazinyl, substituted or unsubstituted pyranyl, substituted or
unsubstituted morpholinyl, substituted or unsubstituted
1,4-dioxanyl, substituted or unsubstituted tetrahydro-2H-pyranyl,
substituted or unsubstituted thianyl, or substituted or
unsubstituted dithianyl. In embodiments, R.sup.2 is substituted or
unsubstituted phenyl, substituted or unsubstituted thiofuranyl,
substituted or unsubstituted imidazolyl, substituted or
unsubstituted pyrazolyl, substituted or unsubstituted triazolyl,
substituted or unsubstituted tetrazolyl, substituted or
unsubstituted furanyl, substituted or unsubstituted oxazolyl,
substituted or unsubstituted isooxazolyl, substituted or
unsubstituted oxadiazolyl, substituted or unsubstituted
oxatriazolyl, substituted or unsubstituted thienyl, substituted or
unsubstituted thiazolyl, substituted or unsubstituted isothiazolyl,
substituted or unsubstituted pyridinyl, substituted or
unsubstituted pyrazinyl, substituted or unsubstituted pyrimidinyl,
substituted or unsubstituted pyridazinyl, or substituted or
unsubstituted triazinyl (e.g., 1,3,5-triazinyl, 1,2,3-triazinyl, or
1,2,4-triazinyl). In embodiments, R.sup.2 is substituted or
unsubstituted indolyl, substituted or unsubstituted benzimidazolyl,
substituted or unsubstituted indazolyl, substituted or
unsubstituted benzotriazolyl, substituted or unsubstituted
pyrrolopyrimidinyl, substituted or unsubstituted purinyl,
substituted or unsubstituted indolizinyl, substituted or
unsubstituted pyrrolopyriazinyl, substituted or unsubstituted
pyrrolopyrimidinyl, substituted or unsubstituted
imidazopyridazinyl, substituted or unsubstituted imidazopyridinyl,
substituted or unsubstituted imidazopyrimidinyl, substituted or
unsubstituted cinnolinyl, substituted or unsubstituted
quinazolinyl, substituted or unsubstituted quinoxalinyl,
substituted or unsubstituted phthalazinyl, substituted or
unsubstituted pyridopyrazinyl, substituted or unsubstituted
pteridinyl, substituted or unsubstituted pyrazolopyridinyl,
substituted or unsubstituted quinolinyl, substituted or
unsubstituted isoquinolinyl, substituted or unsubstituted
naphthyridinyl, or substituted or unsubstituted carbazolyl. In
embodiments, R.sup.2 is substituted aziridinyl, substituted
oxiranyl, substituted thiiranyl, substituted azetidinyl,
substituted oxetanyl, substituted thietanyl, substituted
pyrrolidinyl, substituted pyrrolyl, substituted imidazolyl,
substituted imidazolinyl, substituted pyrazolinyl, substituted
tetrahydrofuranyl, substituted thiolanyl, substituted piperidinyl,
substituted piperazinyl, substituted pyranyl, substituted
morpholinyl, substituted 1,4-dioxanyl, substituted
tetrahydro-2H-pyranyl, substituted thianyl, or substituted
dithianyl. In embodiments, R.sup.2 is substituted phenyl,
substituted thiofuranyl, substituted imidazolyl, substituted
pyrazolyl, substituted triazolyl, substituted tetrazolyl,
substituted furanyl, substituted oxazolyl, substituted isooxazolyl,
substituted oxadiazolyl, substituted oxatriazolyl, substituted
thienyl, substituted thiazolyl, substituted isothiazolyl,
substituted pyridinyl, substituted pyrazinyl, substituted
pyrimidinyl, substituted pyridazinyl, or substituted triazinyl
(e.g., 1,3,5-triazinyl, 1,2,3-triazinyl, or 1,2,4-triazinyl). In
embodiments, R.sup.2 is substituted indolyl, substituted
benzimidazolyl, substituted indazolyl, substituted benzotriazolyl,
substituted pyrrolopyrimidinyl, substituted purinyl, substituted
indolizinyl, substituted pyrrolopyriazinyl, substituted
pynolopyrimidinyl, substituted imidazopyridazinyl, substituted
imidazopyridinyl, substituted imidazopyrimidinyl, substituted
cinnolinyl, substituted quinazolinyl, substituted quinoxalinyl,
substituted phthalazinyl, substituted pyridopyrazinyl, substituted
pteridinyl, substituted pyrazolopyridinyl, substituted quinolinyl,
substituted isoquinolinyl, substituted naphthyridinyl, or
substituted carbazolyl. In embodiments, R.sup.2 is unsubstituted
aziridinyl, unsubstituted oxiranyl, unsubstituted thiiranyl,
unsubstituted azetidinyl, unsubstituted oxetanyl, unsubstituted
thietanyl, unsubstituted pyrrolidinyl, unsubstituted pyrrolyl,
unsubstituted imidazolyl, unsubstituted imidazolinyl, unsubstituted
pyrazolinyl, unsubstituted tetrahydrofuranyl, unsubstituted
thiolanyl, unsubstituted piperidinyl, unsubstituted piperazinyl,
unsubstituted pyranyl, unsubstituted morpholinyl, unsubstituted
1,4-dioxanyl, unsubstituted tetrahydro-2H-pyranyl, unsubstituted
thianyl, or unsubstituted dithianyl. In embodiments, R.sup.2 is
unsubstituted phenyl, unsubstituted thiofuranyl, unsubstituted
imidazolyl, unsubstituted pyrazolyl, unsubstituted triazolyl,
unsubstituted tetrazolyl, unsubstituted furanyl, unsubstituted
oxazolyl, unsubstituted isooxazolyl, unsubstituted oxadiazolyl,
unsubstituted oxatriazolyl, unsubstituted thienyl, unsubstituted
thiazolyl, unsubstituted isothiazolyl, unsubstituted pyridinyl,
unsubstituted pyrazinyl, unsubstituted pyrimidinyl, unsubstituted
pyridazinyl, or unsubstituted triazinyl (e.g., 1,3,5-triazinyl,
1,2,3-triazinyl, or 1,2,4-triazinyl). In embodiments, R.sup.2 is
unsubstituted indolyl, unsubstituted benzimidazolyl, unsubstituted
indazolyl, unsubstituted benzotriazolyl, unsubstituted
pyrrolopyrimidinyl, unsubstituted purinyl, unsubstituted
indolizinyl, unsubstituted pyrrolopyriazinyl, unsubstituted
pyrrolopyrimidinyl, unsubstituted imidazopyridazinyl, unsubstituted
imidazopyridinyl, unsubstituted imidazopyrimidinyl, unsubstituted
cinnolinyl, unsubstituted quinazolinyl, unsubstituted quinoxalinyl,
unsubstituted phthalazinyl, unsubstituted pyridopyrazinyl,
unsubstituted pteridinyl, unsubstituted pyrazolopyridinyl,
unsubstituted quinolinyl, unsubstituted isoquinolinyl,
unsubstituted naphthyridinyl, or unsubstituted carbazolyl.
[0333] In embodiments, --(R.sup.2)-(R.sup.23).sub.z23 is:
##STR00008##
wherein R.sup.23 and z23 are as described herein including
embodiments.
[0334] In embodiments, --(R.sup.2)-(R.sup.23).sub.z23 is:
##STR00009##
wherein R.sup.23 is as described herein, including embodiments.
[0335] In embodiments, R.sup.2 is R.sup.23-substituted phenyl. In
embodiments, R.sup.2 is R.sup.23-substituted 5 to 6 membered
heteroaryl. R.sup.23 is independently halogen, --CX.sup.23.sub.3,
--CHX.sup.23.sub.2, --CH.sub.2X.sup.23, --OCX.sup.23.sub.3,
--OCH.sub.2X.sup.23, --OCHX.sup.22A, --CN, --SO.sub.n23R.sup.100D,
--SO.sub.v23NR.sup.100AR.sup.100B, --NHC(O)NR.sup.100AR.sup.100B,
--NR.sup.100AR.sup.100B, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --N R.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl. Two adjacent
R.sup.23 substituents may optionally be joined to form a
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl. R.sup.100A, R.sup.100B,
R.sup.100C, and R.sup.100D are independently hydrogen, --CX.sub.3,
--CN, --COOH, --CONH.sub.2, --CHX.sub.2, --CH.sub.2X, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl. R.sup.100A and
R.sup.100B substituents bonded to the same nitrogen atom may
optionally be joined to form a substituted or unsubstituted
heterocycloalkyl or substituted or unsubstituted heteroaryl; X and
X.sup.23 are independently --F, --Cl, --Br, or --I. The symbol n23
is independently an integer from 0 to 4. The symbols m23 and v23
are independently 1 or 2.
[0336] In embodiments, R.sup.2A is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.2A is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.2A is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.2A is independently
unsubstituted methyl. In embodiments, R.sup.2A is independently
unsubstituted ethyl. In embodiments, R.sup.2A is independently
unsubstituted propyl. In embodiments, is independently
unsubstituted isopropyl. In embodiments, R.sup.2A is independently
unsubstituted tert-butyl. In embodiments, R.sup.2A is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.2A is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.2A is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.2A is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.2A is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.2A is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.2A is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.2A is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.2A is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.2A is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2A is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.2A is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2A is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered).
[0337] In embodiments, R.sup.2A is independently substituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.2A is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered).
[0338] In embodiments, R.sup.2A is substituted or unsubstituted
aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In
embodiments, R.sup.2A is substituted aryl (e.g., C.sub.6-C.sub.10
aryl, C.sub.10 aryl, or phenyl). In embodiments, R.sup.2A is an
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl).
[0339] In embodiments, R.sup.2B is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.2B is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.2B is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.2B is independently
unsubstituted methyl. In embodiments, R.sup.2B is independently
unsubstituted ethyl. In embodiments, R.sup.2B is independently
unsubstituted propyl. In embodiments, R.sup.2B is independently
unsubstituted isopropyl. In embodiments, R.sup.2B is independently
unsubstituted tert-butyl. In embodiments, R.sup.2B is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.2B is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.2B is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.2B is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.2B is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.2B is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.2B is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.2B is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.2B is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.2B is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2B is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.2B is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2B is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.2B is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.2B is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0340] In embodiments, R.sup.2A and R.sup.2B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.2A and R.sup.2B substituents bonded to the same
nitrogen atom may be joined to form a substituted heterocycloalkyl
(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5
membered, or 5 to 6 membered). In embodiments, R.sup.2A and
R.sup.2B substituents bonded to the same nitrogen atom may be
joined to form an unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered).
[0341] In embodiments, R.sup.2A and R.sup.2B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.2A and R.sup.2B
substituents bonded to the same nitrogen atom may be joined to form
a substituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.2A and R.sup.2B
substituents bonded to the same nitrogen atom may be joined to form
an unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered).
[0342] In embodiments, R.sup.2C is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.2C is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.2C is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.2C is independently
unsubstituted methyl. In embodiments, R.sup.2C is independently
unsubstituted ethyl. In embodiments, R.sup.2C is independently
unsubstituted propyl. In embodiments, R.sup.2C is independently
unsubstituted isopropyl. In embodiments, R.sup.2C is independently
unsubstituted tert-butyl. In embodiments, R.sup.2C is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.2C is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.2C is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.2C is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.2C is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.2C is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.2C is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.2C is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.2C is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.2C is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, is independently
substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl). In
embodiments, R.sup.2C is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2C is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.2C is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.2C is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or S to 6 membered).
[0343] In embodiments, R.sup.2D is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.4,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.2D is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.2D is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R is independently unsubstituted
methyl. In embodiments, R.sup.2D is independently unsubstituted
ethyl. In embodiments, R.sup.2D is independently unsubstituted
propyl. In embodiments, R.sup.2D is independently unsubstituted
isopropyl. In embodiments, R.sup.2D is independently unsubstituted
tert-butyl. In embodiments, R.sup.2D is independently substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered). In
embodiments, R.sup.2D is independently substituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered). In embodiments, R.sup.2D is
independently unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.2D is independently substituted or
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.2D is
independently substituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.2D is independently unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.2D is independently
substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.2D is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.2D is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.2D is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2D is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.2D is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.2D is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.2D is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.2D is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0344] In embodiments, R.sup.2 is independently hydrogen,
halogen, --CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.23-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.23-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.23-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.23-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.23-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.23-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.2 is independently --F,
--Cl, --Br, or --I.
[0345] In embodiments, R.sup.2 is independently hydrogen. In
embodiments, R.sup.2 is independently R.sup.23-substituted methyl.
In embodiments, R.sup.2 is independently R.sup.23-substituted
ethyl. In embodiments, R.sup.2 is independently unsubstituted
methyl. In embodiments, R.sup.2 is independently unsubstituted
ethyl.
[0346] R.sup.23 is independently oxo,
halogen, --CX.sup.23.sub.3, --CHX.sup.23.sub.2, --CH.sub.2X.sup.23,
--OCX.sup.22A, --OCH.sub.2X.sup.23, --OCHX.sup.23.sub.2, --CN,
--OH, --NH.sub.2, --CO OH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.24-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.24-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.24-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.24-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.24-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.24-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.23 is independently oxo, halogen,
--CX.sup.23.sub.3, --CHX.sup.23.sub.2, --CH.sub.2X.sup.23,
--OCX.sup.22A, --OCH.sub.2X.sup.23, --OCHX.sup.22A, --CN, --OH,
--NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H,
--SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.23 is independently --F, --Cl, --Br, or
--I.
[0347] In embodiments, R.sup.23 is halogen or --CX.sup.23.sub.3. In
embodiments, R.sup.23 is independently unsubstituted methyl. In
embodiments, R.sup.23 is independently unsubstituted ethyl. In
embodiments, R.sup.23 is hydrogen. In embodiments, R.sup.23 is
unsubstituted phenyl. In embodiments, R.sup.23 is unsubstituted
thiophenyl. In embodiments, R.sup.23 is R.sup.20-substituted aryl.
In embodiments, R.sup.23 is unsubstituted thienyl. In embodiments,
R.sup.23 is substituted phenyl. In embodiments, R.sup.23 is
R.sup.24-substituted phenyl.
[0348] R.sup.24 is independently oxo,
halogen, --CX.sup.24.sub.3, --CHX.sup.24.sub.2, --CH.sub.2X.sup.24,
--OCX.sup.24.sub.3, --OCH.sub.2X.sup.24, --OCHX.sup.24.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.25-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.3-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.25-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.25-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.25-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.25-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.25-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.24 is independently oxo, halogen,
--CX.sup.24.sub.3, --CHX.sup.24.sub.2, --CH.sub.2X.sup.24,
--OCX.sup.2--OCH.sub.2X.sup.24, --OCHX.sup.24, --CN, --OH,
--NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H,
--SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.24 is independently --F, --Cl, --Br, or
--I.
[0349] In embodiments, R.sup.24 is hydrogen. In embodiments,
R.sup.24 is --OCH.sub.3. In embodiments, R.sup.24 is halogen. In
embodiments, R.sup.24 is --CF.sub.3. In embodiments, R.sup.24 is
--Br. In embodiments, R.sup.24 is --I. In embodiments, R.sup.24 is
unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R.sup.24
is unsubstituted C.sub.1-C.sub.4 alkoxy.
[0350] R.sup.25 is independently oxo,
halogen, --CX.sup.25.sub.3, --CHX.sup.25.sub.2, --CH.sub.2X.sup.25,
--OCX.sup.23.sub.3, --OCH.sub.2X.sup.25, --OCHX.sup.25.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.25 is independently --F,
--Cl, --Br, or --I.
[0351] In embodiments, R.sup.25 is independently unsubstituted
methyl. In embodiments, R.sup.25 is independently unsubstituted
ethyl.
[0352] In embodiments, R.sup.2A is independently hydrogen,
--CX.sup.2A.sub.3, --CHX.sup.2A.sub.2, --CH.sub.2X.sup.2A, --CN,
--COOH, --CONH.sub.2, R.sup.23A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.23A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.23A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.23A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.23A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.23A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2A is independently hydrogen, --CX.sup.2A,
--CHX.sup.2A.sub.2, --CH.sub.2X.sup.2A, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.2A is independently --F,
--Cl, --Br, or --I.
[0353] In embodiments, R.sup.2A is independently hydrogen. In
embodiments, R.sup.2A is independently unsubstituted methyl. In
embodiments, R.sup.2A is independently unsubstituted ethyl.
[0354] In embodiments, R.sup.2A and R.sup.2B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.23A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.23A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2A and R.sup.2B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.2A and R.sup.2B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.23A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.2A and R.sup.2B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0355] R.sup.23A is independently oxo,
halogen, --CX.sup.23A.sub.3, --CHX.sup.23A.sub.2,
--CH.sub.2X.sup.23A, --OCX.sup.23A.sub.3, --OCH.sub.2X.sup.23A,
--OCHX.sup.23A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.24A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.24A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.24A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.24A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.24A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.24A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.23A is independently oxo, halogen,
--CX.sup.23A.sub.3, --CHX.sup.23A.sub.2, --CH.sub.2X.sup.23A,
--OCX.sup.23A.sub.3, --OCH.sub.2X.sup.23A, --OCHX.sup.23A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.23A is independently --F,
--Cl, --Br, or --I.
[0356] In embodiments, R.sup.23A is independently unsubstituted
methyl. In embodiments, R.sup.23A is independently unsubstituted
ethyl.
[0357] R.sup.24A is independently oxo,
halogen, --CX.sup.24A.sub.3, --CHX.sup.24A.sub.2,
--CH.sub.2X.sup.24A, --OCX.sup.24A.sub.3, --OCH.sub.2X.sup.24A,
--OCHX.sup.24A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.25A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.25A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.25A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.25A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.25A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.25A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.24A is independently oxo, halogen,
--CX.sup.24A.sub.3, --CHX.sup.24A.sub.2, --CH.sub.2X.sup.24A,
--OCX.sup.24A.sub.3, --OCH.sub.2X.sup.24A, --OCHX.sup.24A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.24A is independently --F,
--Cl, --Br, or --I.
[0358] R.sup.25A is independently oxo,
halogen, --CX.sup.25A.sub.3, --CHX.sup.25A.sub.2,
--CH.sub.2X.sup.25A, --OCX.sup.25A.sub.3, --OCH.sub.2X.sup.25A,
--OCHX.sup.25A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.25A is independently --F, --Cl, --Br, or --I.
[0359] In embodiments, R.sup.2B is independently hydrogen,
--CX.sup.2B.sub.3, --CHX.sup.2B.sub.2, --CH.sub.2X.sup.B, --CN,
--COOH, --CONH.sub.2, R.sup.23B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.23B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.23B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.23B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.23B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.23B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2B is independently hydrogen, --CX.sup.2B.sub.3,
--CHX.sup.2B.sub.2, --CH.sub.2X.sup.2B, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.2B is independently --F,
--Cl, --Br, or --I.
[0360] In embodiments, R.sup.2B is independently hydrogen. In
embodiments, R.sup.2B is independently unsubstituted methyl. In
embodiments, R.sup.2B is independently unsubstituted ethyl.
[0361] In embodiments, R.sup.2A and R.sup.2B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.23B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.23B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2A and R.sup.2B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.2A and R.sup.2B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.23B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.2A and R.sup.2B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0362] R.sup.23B is independently oxo,
halogen, --CX.sup.23B.sub.3, --CHX.sup.23B.sub.2,
--CH.sub.2X.sup.23B, --OCX.sup.23B.sub.3, --OCH.sub.2X.sup.238,
--OCHX.sup.23B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.24B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.24B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.24B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.24B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.24B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.24B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.23B is independently oxo, halogen,
--CX.sup.23B.sub.3, --CHX.sup.23B.sub.2, --CH.sub.2X.sup.238,
--OCX.sup.23B.sub.3, --OCH.sub.2X.sup.23B, --OCHX.sup.23B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.23B is independently --F,
--Cl, --Br, or --I.
[0363] In embodiments, R.sup.23B is independently unsubstituted
methyl. In embodiments, R.sup.238 is independently unsubstituted
ethyl.
[0364] R.sup.24B is independently oxo,
halogen, --CX.sup.24B.sub.3, --CHX.sup.24B.sub.2,
--CH.sub.2X.sup.24B, --OCX.sup.24B.sub.3, --OCH.sub.2X.sup.24B,
--OCHX.sup.24B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.25B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.25B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.25B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.25B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.25B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.25B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.24B is independently oxo, halogen,
--CX.sup.24B.sub.3, --CHX.sup.24B.sub.2, --CH.sub.2X.sup.24B,
--OCX.sup.24B.sub.3, --OCH.sub.2X.sup.24B, --OCHX.sup.24B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.24B is independently --F,
--Cl, --Br, or --I.
[0365] R.sup.25B is independently oxo,
halogen, --CX.sup.25B.sub.3, --CHX.sup.25B.sub.2,
--CH.sub.2X.sup.25B, --OCX.sup.25B.sub.3, --OCH.sub.2X.sup.25B,
--OCHX.sup.25B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.25B is independently --F, --Cl, --Br, or --I.
[0366] In embodiments, R.sup.2C is independently hydrogen,
--CX.sup.2C.sub.3, --CHX.sup.2C.sub.2, --CH.sub.2X.sup.2C, --CN,
--COOH, --CONH.sub.2, R.sup.23C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.23C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.23C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.23C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.23C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.23C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2C is independently hydrogen, --CX.sup.2C.sub.3,
--CHX.sup.2C.sub.2, --CH.sub.2X.sup.2C, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.2C is independently --F,
--Cl, --Br, or --I.
[0367] In embodiments, R.sup.2C is independently hydrogen. In
embodiments, R.sup.2C is independently unsubstituted methyl. In
embodiments, R.sup.2C is independently unsubstituted ethyl.
[0368] R.sup.23D is independently oxo,
halogen, --CX.sup.23C.sub.3, --CHX.sup.23C.sub.2,
--CH.sub.2X.sup.23C, --OCX.sup.23C.sub.3, --OCH.sub.2X.sup.23C,
--OCHX.sup.23C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.24C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.24C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.24C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.24C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.24C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.24C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.23C is independently oxo. halogen,
--CX.sup.23C.sub.3, --CHX.sup.23C.sub.2, --CH.sub.2X.sup.23C,
--OCX.sup.23C.sub.3, --OCH.sub.2X.sup.23C, --OCHX.sup.23C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.1-C.sub.4, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.23C is independently --F,
--Cl, --Br, or --I.
[0369] In embodiments, R.sup.23D is independently unsubstituted
methyl. In embodiments, R.sup.23C is independently unsubstituted
ethyl.
[0370] R.sup.24C is independently oxo,
halogen, --CX.sup.24C.sub.3, --CHX.sup.24C.sub.2,
--CH.sub.2X.sup.24C, --OCX.sup.24C.sub.3, --OCH.sub.2X.sup.24C,
--OCHX.sup.24C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.25C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.25C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.25C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.25C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.25C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.25C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.24C is independently oxo, halogen,
--CX.sup.24C.sub.3, --CHX.sup.24C.sub.2, --CH.sub.2X.sup.24C,
--OCX.sup.24C.sub.3, --OCH.sub.2X.sup.24C, --OCHX.sup.24C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.24C is independently --F,
--Cl, --Br, or --I.
[0371] R.sup.25C is independently oxo,
halogen, --CX.sup.25C.sub.3, --CHX.sup.25C.sub.2,
--CH.sub.2X.sup.25C, --OCX.sup.25C.sub.3, --OCH.sub.2X.sup.25C,
--OCHX.sup.25C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.25C is independently --F, --Cl, --Br, or --I.
[0372] In embodiments, R.sup.2D is independently hydrogen,
--CX.sup.2D.sub.3, --CHX.sup.2D.sub.2, --CH.sub.2X.sup.2D, --CN,
--COOH, --CONH.sub.2, R.sup.23D-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.23D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.23D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.23D-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.23D-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.2D is independently hydrogen, --CX.sup.2D.sub.3,
--CHX.sup.2D.sub.2, --CH.sub.2X.sup.2D, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.2D is independently --F,
--Cl, --Br, or --I.
[0373] In embodiments, R.sup.2D is independently hydrogen. In
embodiments, R.sup.2D is independently unsubstituted methyl. In
embodiments, R.sup.2D is independently unsubstituted ethyl.
[0374] R.sup.23D is independently oxo,
halogen, --CX.sup.23D.sub.3, --CHX.sup.23D.sub.2,
--CH.sub.2X.sup.23D, --OCX.sup.23D.sub.3, --OCH.sub.2X.sup.23D,
--OCHX.sup.23D.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.24D-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.24D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.24D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.24D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.24D-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.24D-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.23D is independently oxo, halogen,
--CX.sup.23D.sub.3, --CHX.sup.23D.sub.2, --CH.sub.2X.sup.23D,
--OCX.sup.23D.sub.3, --OCH.sub.2X.sup.23D, --OCHX.sup.23D.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.23D is independently --F,
--Cl, --Br, or --I.
[0375] In embodiments, R.sup.23D is independently unsubstituted
methyl. In embodiments, R.sup.23D is independently unsubstituted
ethyl.
[0376] R.sup.24D is independently oxo,
halogen, --CX.sup.24D.sub.3, --CHX.sup.24D.sub.2,
--CH.sub.2X.sup.24D, --OCX.sup.24D.sub.3, --OCH.sub.2X.sup.24D,
--OCHX.sup.24D.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.25D-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.25D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.25D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.25D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.25D-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.25D-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.24D is independently oxo, halogen,
--CX.sup.24D.sub.3, --CHX.sup.24D.sub.2, --CH.sub.2X.sup.24D,
--OCX.sup.24D.sub.3, --OCH.sub.2X.sup.24D, --OCHX.sup.24D.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.24D is independently --F,
--Cl, --Br, or --I.
[0377] R.sup.25D is independently oxo,
halogen, --CX.sup.25D.sub.3, --CHX.sup.25D.sub.2,
--CH.sub.2X.sup.23D, --OCX.sup.25D.sub.3, --OCH.sub.2X.sup.25D,
--OCHX.sup.25D.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.25D is independently --F, --Cl, --Br, or --I.
[0378] In embodiments, R.sup.3 is independently hydrogen,
--CX.sup.3.sub.3, --CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN,
--C(O)R.sup.3C, --C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B,
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0379] In embodiments, R.sup.3 is independently hydrogen,
--CX.sup.3.sub.3, --CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN,
--C(O)R.sup.3C, --C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B,
substituted or unsubstituted C.sub.1-C.sub.4 alkyl, or substituted
or unsubstituted 2 to 4 membered heteroalkyl, substituted or
unsubstituted C.sub.3-C.sub.6 cycloalkyl, substituted or
unsubstituted 3 to 6 membered heterocycloalkyl, substituted or
unsubstituted phenyl, or substituted or unsubstituted 5 to 6
membered heteroaryl. In embodiments, R.sup.3 is independently
hydrogen, --CX.sup.3.sub.3, --CHX.sup.3.sub.2, --CH.sub.2X.sup.3,
--CN, --C(O)R.sup.3C, --C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B,
unsubstituted C.sub.1-C.sub.4 alkyl, or unsubstituted 2 to 4
membered heteroalkyl.
[0380] In embodiments, R.sup.3A is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.3A is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.3A is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.3A is independently
unsubstituted methyl. In embodiments, R.sup.3A is independently
unsubstituted ethyl. In embodiments, R.sup.3A is independently
unsubstituted propyl. In embodiments, R.sup.3A is independently
unsubstituted isopropyl. In embodiments, R.sup.3A is independently
unsubstituted tert-butyl. In embodiments, R.sup.3A is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.3A is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered). In embodiments,
R.sup.3A is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.3A is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.3A is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.3A is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.3A is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.A is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.3A is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.3A is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.3A is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.3A is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.3A is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.3A is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.3A is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0381] In embodiments, R.sup.3B is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.3B is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.3B is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.3B is independently
unsubstituted methyl. In embodiments, R.sup.3B is independently
unsubstituted ethyl. In embodiments, R.sup.3B is independently
unsubstituted propyl. In embodiments, R.sup.3B is independently
unsubstituted isopropyl. In embodiments, R.sup.3B is independently
unsubstituted tert-butyl. In embodiments, R.sup.3B is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.3B is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.3B is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.3B is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.3B is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.3B is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.3B is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.3B is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.3B is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.3B is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.3B is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.3B is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.3B is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.3B is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.3B is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0382] In embodiments, R.sup.3A and R.sup.3B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.3A and R.sup.3B substituents bonded to the same
nitrogen atom may be joined to form a substituted heterocycloalkyl
(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5
membered, or 5 to 6 membered). In embodiments, R.sup.3A and
R.sup.3B substituents bonded to the same nitrogen atom may be
joined to form an unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered).
[0383] In embodiments, R.sup.3A and R.sup.3B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.3A and R.sup.3B
substituents bonded to the same nitrogen atom may be joined to form
a substituted heteroaryl (e.g., 5 to 10 membered, S to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.3A and R.sup.3B
substituents bonded to the same nitrogen atom may be joined to form
an unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered).
[0384] In embodiments, R.sup.3C is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.3C is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.3C is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.30 is independently
unsubstituted methyl. In embodiments, R.sup.3C is independently
unsubstituted ethyl. In embodiments, R.sup.3C is independently
unsubstituted propyl. In embodiments, R.sup.3C is independently
unsubstituted isopropyl. In embodiments, R.sup.3C is independently
unsubstituted tert-butyl. In embodiments, R.sup.3C is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 3
membered). In embodiments, R.sup.3C is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered). In embodiments,
R.sup.3C is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.3C is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.3C is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.3C is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.3C is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.3C is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.3C is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.3C is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.30 is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.3C is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.3C is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.3C is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.3C is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0385] In embodiments, R.sup.3 is independently hydrogen,
--CX.sup.3.sub.3, --CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN,
C(O)R.sup.3C, --C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B,
R.sup.26-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.26-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.26-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.26-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.26-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.26-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.6, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.3 is independently --F,
--Cl, --Br, or --I.
[0386] In embodiments, R.sup.3 is independently hydrogen. In
embodiments, R.sup.3 is independently unsubstituted methyl. In
embodiments, R.sup.3 is independently unsubstituted ethyl.
[0387] R.sup.26 is independently oxo,
halogen, --CX.sup.26.sub.3, --CHX.sup.26.sub.2, --CH.sub.2X.sup.26,
--OCX.sup.26.sub.3, --OCH.sub.2X.sup.26, --OCHX.sup.26.sub.2, --CN,
--OH, --NH.sub.2, --CO OH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.27-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.27-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.27-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.27-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.27-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.27-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.26 is independently oxo, halogen,
--CX.sup.26.sub.3, --CHX.sup.26.sub.2, --CH.sub.2X.sup.26,
--OCX.sup.26.sub.3, --OCH.sub.2X.sup.26, --OCHX.sup.26.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.26 is independently --F,
--Cl, --Br, or --I.
[0388] In embodiments, R.sup.26 is independently unsubstituted
methyl. In embodiments, R.sup.26 is independently unsubstituted
ethyl.
[0389] R.sup.27 is independently oxo,
halogen, --CX.sup.27.sub.3, --CHX.sup.27.sub.2, --CH.sub.2X.sup.27,
--OCX.sup.27.sub.3, --OCH.sub.2X.sup.27, --OCHX.sup.27.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.28-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.28-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 3 membered), R.sup.28-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.28-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.28-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.28-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.27 is independently oxo, halogen,
--CX.sup.27.sub.3, --CHX.sup.27.sub.2, --CH.sub.2X.sup.27,
--OCX.sup.27.sub.3, --OCH.sub.2X.sup.27, --OCHX.sup.27.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.27 is independently --F,
--Cl, --Br, or --I. Ln embodiments, R.sup.27 is independently
unsubstituted methyl. In embodiments, R.sup.27 is independently
unsubstituted ethyl.
[0390] R.sup.2B is independently oxo,
halogen, --CX.sup.28.sub.3, --CHX.sup.28.sub.2, --CH.sub.2X.sup.28,
--OCX.sup.28.sub.3, --OCH.sub.2X.sup.28, --OCHX.sup.28.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.28 is independently --F,
--Cl, --Br, or --I.
[0391] In embodiments, R.sup.2B is independently unsubstituted
methyl. In embodiments, R.sup.2B is independently unsubstituted
ethyl.
[0392] In embodiments, R.sup.3A is independently hydrogen,
--CX.sup.3A.sub.3, --CHX.sup.3A.sub.2, --CH.sub.2X.sup.3A, --CN,
--COOH, --CONH.sub.2, R.sup.26A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.26A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.26A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.26A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.26A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.20-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3A is independently hydrogen, --CX.sup.3A.sub.3,
--CHX.sup.3A.sub.2, --CH.sub.2X.sup.3A, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.3A is independently --F,
--Cl, --Br or --I.
[0393] In embodiments, R.sup.3A is independently hydrogen. In
embodiments, R.sup.3A is independently unsubstituted methyl. In
embodiments, R.sup.3A is independently unsubstituted ethyl.
[0394] In embodiments, R.sup.3A and R.sup.3B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.26A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.26A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3A and R.sup.3B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.3A and R.sup.3B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.26A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.3A and R.sup.3B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0395] R.sup.26A is independently oxo,
halogen, --CX.sup.22A.sub.3, --CHX.sup.26A.sub.2,
--CH.sub.2X.sup.22A, --OCX.sup.26A.sub.3, --OCH.sub.2X.sup.22A,
--OCHX.sup.26A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.27A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.27A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.27A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.27A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.27A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), R.sup.27A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.20A is independently oxo, halogen,
--CX.sup.26A.sub.3, --CHX.sup.26A.sub.2, --CH.sub.2X.sup.26A,
--OCX.sup.26A.sub.3, --OCH.sub.2X.sup.26A, --OCHX.sup.26A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered). X.sup.26A is independently --F,
--Cl, --Br, or --I.
[0396] In embodiments, R.sup.26A is independently unsubstituted
methyl. In embodiments, R.sup.26A is independently unsubstituted
ethyl.
[0397] R.sup.27A is independently oxo,
halogen, --CX.sup.27A.sub.3, --CHX.sup.27A.sub.2,
--CH.sub.2X.sup.27A, --OCX.sup.27A.sub.3, --OCH.sub.2X.sup.27A,
--OCHX.sup.27A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.20-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.20-substituted or unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), R.sup.28A-substituted or
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), R.sup.28A-substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.28A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.28A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.27A is independently oxo, halogen,
--CX.sup.27A.sub.3, --CHX.sup.27A.sub.2, --CH.sub.2X.sup.27A,
--OCX.sup.27A.sub.3, --OCH.sub.2X.sup.27A, --OCHX.sup.27A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.27A is independently --F,
--Cl, --Br, or --I.
[0398] R.sup.28A is independently oxo,
halogen, --CX.sup.28A.sub.3, --CHX.sup.28A.sub.2,
--CH.sub.2X.sup.28A, --OCX.sup.28A.sub.3, --OCH.sub.2X.sup.28A,
--OCHX.sup.28A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.28A is independently --F, --Cl, --Br, or --I.
[0399] In embodiments, R.sup.3B is independently hydrogen,
--CX.sup.3B.sub.3, --CHX3.sup.B.sub.2, --CH.sub.2X.sup.3B, --CN,
--COOH, --CONH.sub.2, R.sup.26B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.20-substituted or unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), R.sup.26B-substituted or
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), R.sup.20-substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.26B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.26B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3B is independently hydrogen, --CX.sup.3B.sub.3,
--CHX.sup.3B.sub.2, --CH.sub.2X.sup.3B, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.3B is independently --F,
--Cl, --Br, or --I.
[0400] In embodiments, R.sup.3B is independently hydrogen. In
embodiments, R.sup.3B is independently unsubstituted methyl. In
embodiments, R.sup.3B is independently unsubstituted ethyl.
[0401] In embodiments, R.sup.3A and R.sup.3B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.26B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.26B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3A and R.sup.3B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.3A and R.sup.3B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.26B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.3A and R.sup.3B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0402] R.sup.26B is independently oxo,
halogen, --CX.sup.26B.sub.3, --CHX.sup.26B.sub.2,
--CH.sub.2X.sup.26B, --OCX.sup.26B.sub.3, --OCH.sub.2X.sup.26B,
--OCHX.sup.26B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.27B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.27B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered),
R.sup.27B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.27B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.27B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), R.sup.27B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.26B is independently oxo, halogen,
--CX.sup.26--CHX.sup.26B.sub.2, --CH.sub.2X.sup.26B,
--OCX.sup.26B.sub.3, --OCH.sub.2X.sup.26B, --OCHX.sup.2.RTM..sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered). X.sup.26B is independently --F,
--Cl, --Br, or --I.
[0403] In embodiments, R.sup.26B is independently unsubstituted
methyl. In embodiments, R.sup.26B is independently unsubstituted
ethyl.
[0404] R.sup.27B is independently oxo,
halogen, --CX.sup.27B.sub.3, --CHX.sup.27B.sub.2,
--CH.sub.2X.sup.27B, --OCX.sup.27B.sub.3, --OCHX.sup.27B,
--OCHX.sup.27B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.28B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.28B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered),
R.sup.28B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.28B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.28B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.28B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.27B is independently oxo, halogen,
--CX.sup.27B.sub.3, --CHX.sup.27B.sub.2, --CH.sub.2X.sup.27B,
--OCX.sup.27B.sub.3, --OCH.sub.2X.sup.273, --OCHX.sup.27B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.27B is independently --F,
--Cl, --Br, or --I.
[0405] R.sup.28B is independently oxo,
halogen, --CX.sup.28B.sub.3, --CHX.sup.28B.sub.2,
--CH.sub.2X.sup.288, --OCX.sup.28B.sub.3, --OCH.sub.2X.sup.28B,
--OCHX.sup.28B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 3 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.28B is independently --F, --Cl, --Br, or --I.
[0406] In embodiments, R.sup.3C is independently hydrogen,
--CX.sup.3C.sub.3, --CHX.sup.3C.sub.2, --CH.sub.2X.sup.3C, --CN,
--COOH, --CONH.sub.2, R.sup.26C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.26C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.26C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.26C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.26C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.26C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3C is independently hydrogen, --CX.sup.3C.sub.3,
--CHX.sup.3C.sub.2, --CH.sub.2X.sup.3C, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.3C is independently --F,
--Cl, --Br, or --I.
[0407] In embodiments, R.sup.3C is independently hydrogen. In
embodiments, R.sup.3C is independently unsubstituted methyl. In
embodiments, R.sup.3C is independently unsubstituted ethyl.
[0408] R.sup.26C is independently oxo,
halogen, --CX.sup.26C.sub.3, --CHX.sup.26C.sub.2,
--CH.sub.2X.sup.26C, --OCX.sup.26C.sub.3, --OCH.sub.2X.sup.26C,
--OCHX.sup.26C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.27C-substituted unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.27C-substituted unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), R.sup.27C-substituted unsubstituted
cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), R.sup.27C-substituted
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.27C-substituted unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), R.sup.27C-substituted or unsubstituted heteroaryl (e.g., 5
to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.26C is independently oxo, halogen,
--CX.sup.26C.sub.3, --CHX.sup.26C.sub.2, --CH.sub.2X.sup.26C,
--OCX.sup.26C.sub.3, --OCH.sub.2X.sup.26C, --OCHX.sup.26C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered). X.sup.26C is independently --F,
--Cl, --Br, or --I.
[0409] In embodiments, R.sup.26C is independently unsubstituted
methyl. In embodiments, R.sup.26C is independently unsubstituted
ethyl.
[0410] R.sup.27C is independently oxo,
halogen, --CX.sup.27C.sub.3, --CHX.sup.27C.sub.2,
--CH.sub.2X.sup.270, --OCX.sup.27C.sub.3, --OCH.sub.2X.sup.27C,
--OCHX.sup.27C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.28C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.28C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.28C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.28C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.28C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.28C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.27C is independently oxo, halogen,
--CX.sup.27C.sub.3, --CHX.sup.27C.sub.2, --CH.sub.2X.sup.27C,
--OCX.sup.27C.sub.3, --OCH.sub.2X.sup.270, --OCHX.sup.27C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.27C is independently --F,
--Cl, --Br, or --I.
[0411] R.sup.28C is independently oxo,
halogen, --CX.sup.28C.sub.3, --CHX.sup.28C.sub.2,
--CH.sub.2X.sup.28C, --OCX.sup.28C.sub.3, --OCH.sub.2X.sup.28C,
--OCHX.sup.28C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.28C is independently --F, --Cl, --Br, or --I.
[0412] In embodiments, R.sup.4 is independently hydrogen,
--CX.sup.4.sub.3, --CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN,
--C(O)R.sup.4C, --C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B,
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0413] In embodiments, R.sup.4 is independently hydrogen,
halogen, --CX.sup.4.sub.3, --CN, --CHX.sup.4.sub.2,
--CH.sub.2X.sup.4, --COOH, --CONH.sub.2, substituted or
unsubstituted C.sub.1-C.sub.4 alkyl, or substituted or
unsubstituted 2 to 4 membered heteroalkyl, substituted or
unsubstituted C.sub.3-C.sub.6 cycloalkyl, substituted or
unsubstituted 3 to 6 membered heterocycloalkyl, substituted or
unsubstituted phenyl, or substituted or unsubstituted 5 to 6
membered heteroaryl. In embodiments, R.sup.4 is independently
hydrogen, halogen, --CX.sup.4.sub.3, --CN, --CHX.sup.4.sub.2,
--CH.sub.2X.sup.4, unsubstituted C.sub.1-C.sub.4 alkyl, or
unsubstituted 2 to 4 membered heteroalkyl.
[0414] In embodiments, R.sup.4A is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.4A is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.4A is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.4A is independently
unsubstituted methyl. In embodiments, R.sup.4A is independently
unsubstituted ethyl. In embodiments, R.sup.4A is independently
unsubstituted propyl. In embodiments, R.sup.4A is independently
unsubstituted isopropyl. In embodiments, R.sup.4A is independently
unsubstituted tert-butyl. In embodiments, R.sup.4A is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 3
membered). In embodiments, R.sup.4A is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered). In embodiments,
R.sup.4A is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 3 membered). In embodiments, R.sup.4A is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.4A is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.4A is independently unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.4A is independently
substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.A is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.4A is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.4A is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.4A is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.4A is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.4A is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.4A is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.4A is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0415] In embodiments, R.sup.4B is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.4B is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.4B is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.4B is independently
unsubstituted methyl. In embodiments, R.sup.4B is independently
unsubstituted ethyl. In embodiments, R.sup.4B is independently
unsubstituted propyl. In embodiments, R.sup.4B is independently
unsubstituted isopropyl. In embodiments, R.sup.4B is independently
unsubstituted tert-butyl. In embodiments, R.sup.4B is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 3
membered). In embodiments, R.sup.4B is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered). In embodiments,
R.sup.4B is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.4B is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.4B is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.4B is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.4, or C.sub.5-C.sub.6). In embodiments, R.sup.4B is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.4B is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.4B is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.4B is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.4B is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.4B is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.4B is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.4B is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.4B is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0416] In embodiments, R.sup.4A and R.sup.4B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.4A and R.sup.4B substituents bonded to the same
nitrogen atom may be joined to form a substituted heterocycloalkyl
(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5
membered, or 5 to 6 membered). In embodiments, R.sup.4A and
R.sup.4B substituents bonded to the same nitrogen atom may be
joined to form an unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered).
[0417] In embodiments, R.sup.4A and R.sup.4B substituents bonded to
the same nitrogen atom may be joined to form a substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.4A and R.sup.4B
substituents bonded to the same nitrogen atom may be joined to form
a substituted heteroaryl (e.g., 5 to 10 membered, 3 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.4A and R.sup.4B
substituents bonded to the same nitrogen atom may be joined to form
an unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered).
[0418] In embodiments, R.sup.4C is independently substituted or
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In embodiments, R.sup.4C is
independently substituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2). In
embodiments, R.sup.4C is independently unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2). In embodiments, R.sup.4C is independently
unsubstituted methyl. In embodiments, R.sup.4C is independently
unsubstituted ethyl. In embodiments, R.sup.4C is independently
unsubstituted propyl. In embodiments, R.sup.4C is independently
unsubstituted isopropyl. In embodiments, R.sup.4C is independently
unsubstituted tert-butyl. In embodiments, R.sup.4C is independently
substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2
to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered). In embodiments, R.sup.4C is independently substituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered). In embodiments,
R.sup.4C is independently unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered). In embodiments, R.sup.4C is independently
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R.sup.4C is independently substituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R.sup.4C is independently
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R.sup.4C is
independently substituted or unsubstituted heterocycloalkyl (e.g.,
3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered). In embodiments, R.sup.4C is independently
substituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered). In
embodiments, R.sup.4C is independently unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.4C is independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.4C is
independently substituted aryl (e.g., C.sub.6-C.sub.10 or phenyl).
In embodiments, R.sup.4C is independently unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl). In embodiments, R.sup.4C is
independently substituted or unsubstituted heteroaryl (e.g., 5 to
10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.4C is independently substituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered). In embodiments,
R.sup.4C is independently unsubstituted heteroaryl (e.g., 5 to 10
membered, 5 to 9 membered, or 5 to 6 membered).
[0419] In embodiments, R.sup.4 is independently hydrogen,
--CX.sup.4.sub.3, --CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN,
--C(O)R.sup.4C, --C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B,
R.sup.29-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.29-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.29-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.29-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.29-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.29-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.4 is independently hydrogen, --CX.sup.4.sub.3,
--CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN, --C(O)R.sup.4C,
--C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B, unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.1-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.4 is independently --F, --Cl, --Br, or
--I.
[0420] In embodiments, R.sup.4 is independently hydrogen. In
embodiments, R.sup.4 is independently unsubstituted methyl. In
embodiments, R.sup.4 is independently unsubstituted ethyl.
[0421] R.sup.29 is independently oxo,
halogen, --CX.sup.29.sub.3, --CHX.sup.29.sub.2, --CH.sub.2X.sup.29,
--OCX.sup.29.sub.3, --OCH.sub.2X.sup.29, --OCHX.sup.22A, --CN,
--OH, --NH.sub.2, --CO OH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.KO)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.30-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.30-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.30-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.30-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.30-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.30-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.29 is independently oxo, halogen,
--CX.sup.29.sub.3, --CHX.sup.29.sub.2, --CH.sub.2X.sup.29,
--OCX.sup.29.sub.3, --OCH.sub.2X.sup.29, --OCHX.sup.29.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.29 is independently --F,
--Cl, --Br, or --I.
[0422] In embodiments, R.sup.29 is independently unsubstituted
methyl. In embodiments, R.sup.29 is independently unsubstituted
ethyl.
[0423] R.sup.30 is independently oxo,
halogen, --CX.sup.30.sub.3, --CHX.sup.30.sub.2, --CH.sub.2X.sup.30,
--OCX.sup.30.sub.3, --OCH.sub.2X.sup.30, --OCHX.sup.30.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.31-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.31-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.31-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.31-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.31-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.31-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.30 is independently oxo, halogen,
--CX.sup.30.sub.3, --CHX.sup.30.sub.2, --CH.sub.2X.sup.30,
--OCX.sup.30--, --OCH.sub.2X.sup.30, --OCHX.sup.30.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.1-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.30 is independently --F,
--Cl, --Br, or --I. In embodiments, R.sup.30 is independently
unsubstituted methyl. In embodiments, R.sup.30 is independently
unsubstituted ethyl.
[0424] R.sup.31 is independently oxo,
halogen, --CX.sup.31.sub.3, --CHX.sup.31.sub.2, --CH.sub.2X.sup.31,
--OCX.sup.31.sub.3, --OCH.sub.2X.sup.31, --OCHX.sup.31.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.3NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.3' is independently --F,
--Cl, --Br, or --I.
[0425] In embodiments, R.sup.31 is independently unsubstituted
methyl. In embodiments, R.sup.31 is independently unsubstituted
ethyl.
[0426] In embodiments, R.sup.4A is independently hydrogen,
--CX.sup.4A.sub.3, --CHX.sup.4A.sub.2, --CH.sub.2X.sup.4A, --CN,
--COOH, --CONH.sub.2, R.sup.29A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.29A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.29A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.29A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.29A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.29A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.4A is independently hydrogen, --CX.sup.4A.sub.3,
--CHX.sup.4A.sub.2, --CH.sub.2X.sup.4A, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.4A is independently --F,
--Cl, --Br, or --I. In embodiments, R.sup.4A is independently
hydrogen. In embodiments, R.sup.4A is independently unsubstituted
methyl. In embodiments, R.sup.4A is independently unsubstituted
ethyl.
[0427] In embodiments, R.sup.4A and R.sup.4B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.29A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.29A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.4A and R.sup.4B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.4A and R.sup.4B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.29A-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.4A and R.sup.4B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0428] R.sup.20A is independently oxo,
halogen, --CX.sup.29A.sub.3, --CHX.sup.29A.sub.2,
--CH.sub.2X.sup.29A, --OCX.sup.29A.sub.3, --OCH.sub.2X.sup.29A,
--OCHX.sup.29A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.30A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.30A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.30A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.30A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.30A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.30A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.22A is independently oxo, halogen,
--CX.sup.29A.sub.3, --CHX.sup.29A.sub.2, --CH.sub.2X.sup.22A,
--OCX.sup.29A.sub.3, --OCH.sub.2X.sup.29A, --OCHX.sup.29A.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.29A is independently --F,
--Cl, --Br, or --I.
[0429] In embodiments, R.sup.29A is independently unsubstituted
methyl. In embodiments, R.sup.20A is independently unsubstituted
ethyl.
[0430] R.sup.20A is independently oxo,
halogen, --CX.sup.30A.sub.3, --CHX.sup.30A.sub.2,
--CH.sub.2X.sup.30A, --OCX.sup.30A.sub.3, --OCH.sub.2X.sup.30A,
--OCHX.sup.30A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.31A-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.31A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.31A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.31A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.31A-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.31A-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.30A is independently oxo, halogen,
--CX.sup.30A.sub.3, --CHX.sup.30A.sub.2, --CH.sub.2X.sup.30A,
--OCX.sup.30A, --OCH.sub.2X.sup.30A, --OCHX.sup.30A.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.30A is independently --F,
--Cl, --Br, or --I.
[0431] R.sup.31A is independently oxo,
halogen, --CX.sup.31A.sub.3, --CHX.sup.31A.sub.2,
--CH.sub.2X.sup.31A, --OCX.sup.31A.sub.3, --OCH.sub.2X.sup.31A,
--OCHX.sup.31A.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.31A is independently --F, --Cl, --Br, or --I
[0432] In embodiments, R.sup.4B is independently hydrogen,
--CX.sup.4B.sub.3, --CHX.sup.4B.sub.2, --CH.sub.2X.sup.4B, --CN,
--COOH, --CONH.sub.2, R.sup.29B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.29B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.29B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.29B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.29B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.29B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.4B is independently hydrogen, --CX.sup.4B.sub.3,
--CHX.sup.4B.sub.2, --CH.sub.2X.sup.4B, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.4B is independently --F,
--Cl, --Br, or --I.
[0433] In embodiments, R.sup.4B is independently hydrogen. In
embodiments, R.sup.4B is independently unsubstituted methyl. In
embodiments, R.sup.4B is independently unsubstituted ethyl.
[0434] In embodiments, R.sup.4A and R.sup.4B substituents bonded to
the same nitrogen atom may optionally be joined to form a
R.sup.29B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered) or R.sup.29B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.4A and R.sup.4B substituents bonded to the same
nitrogen atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered) or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.4A and R.sup.4B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.29B-substituted or unsubstituted heterocycloalkyl (e.g., 3 to
8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered). In embodiments, R.sup.4A and R.sup.4B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6
membered).
[0435] R.sup.29B is independently oxo,
halogen, --CX.sup.29B.sub.3, --CHX.sup.29B.sub.2,
--CH.sub.2X.sup.29B, --OCX.sup.29B.sub.3, --OCH.sub.2X.sup.29B,
--OCHX.sup.29B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.30B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.30B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.30B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.30B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.30B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.30B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.29B is independently oxo, halogen,
--CX.sup.29B.sub.3, --CHX.sup.29B.sub.2, --CH.sub.2X.sup.29B,
--OCX.sup.29B.sub.3, --OCH.sub.2X.sup.29B, --OCHX.sup.29B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.1-C.sub.4, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.29B is independently --F,
--Cl, --Br, or --I.
[0436] In embodiments, R.sup.29B is independently unsubstituted
methyl. In embodiments, R.sup.29B is independently unsubstituted
ethyl.
[0437] R.sup.30B is independently oxo,
halogen, --CX.sup.30B.sub.3, --CHX.sup.30B.sub.2,
--CH.sub.2X.sup.30B, --OCX.sup.30B.sub.3, --OCH.sub.2X.sup.30B,
--OCHX.sup.30B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.31B-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.31B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.31B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.31B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.31B-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.31B-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.30B is independently oxo, halogen,
--CX.sup.30B.sub.3, --CHX.sup.30B.sub.2, --CH.sub.2X.sup.30B,
--OCX.sup.30B, --OCH.sub.2X.sup.30B, --OCHX.sup.30B.sub.2, --CN,
--OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.30B is independently --F,
--Cl, --Br, or --I.
[0438] R.sup.31B is independently oxo,
halogen, --CX.sup.31B.sub.3, --CHX.sup.31B.sub.2,
--CH.sub.2X.sup.31B, --OCX.sup.31B.sub.3, --OCH.sub.2X.sup.31B,
--OCHX.sup.31B.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.31B is independently --F, --Cl, --Br, or --I
[0439] In embodiments, R.sup.40 is independently hydrogen,
--CX.sup.4C.sub.3, --CHX.sup.4C.sub.2, --CH.sub.2X.sup.4C, --CN,
--COOH, --CONH.sub.2, R.sup.29C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.29C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.29C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.29C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.29C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.29C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.4C is independently hydrogen, --CX.sup.4C.sub.3,
--CHX.sup.4C.sub.2, --CH.sub.2X.sup.4C, --CN, --COOH, --CONH.sub.2,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.4C is independently --F,
--Cl, --Br, or --I. In embodiments, R.sup.4C is independently
hydrogen. In embodiments, R.sup.4C is independently unsubstituted
methyl. In embodiments, R.sup.4C is independently unsubstituted
ethyl.
[0440] R.sup.29C is independently oxo,
halogen, --CX.sup.29C.sub.3, --CHX.sup.29C.sub.2,
--CH.sub.2X.sup.29C, --OCX.sup.29C.sub.3, --OCH.sub.2X.sup.29C,
--OCHX.sup.29C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.30C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.30C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.30C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.30C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.30C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.290 is independently oxo, halogen,
--CX.sup.29C.sub.3, --CHX.sup.29C.sub.2, --CH.sub.2X.sup.29C,
--OCX.sup.29C.sub.3, --OCH.sub.2X.sup.290, --OCHX.sup.29C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.29C is independently --F,
--Cl, --Br, or --I.
[0441] In embodiments, R.sup.29C is independently unsubstituted
methyl. In embodiments, R.sup.29C is independently unsubstituted
ethyl.
[0442] R.sup.30C is independently oxo,
halogen, --CX.sup.30C.sub.3, --CHX.sup.30C.sub.2,
--CH.sub.2X.sup.30C, --OCX.sup.30C.sub.3, --OCH.sub.2X.sup.30C,
--OCHX.sup.30C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, R.sup.31C-substituted or unsubstituted alkyl
(e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.31C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 3 membered),
R.sup.31C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.31C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.31C-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.31C-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.30C is independently oxo, halogen,
--CX.sup.30C.sub.3, --CHX.sup.30C.sub.2, --CH.sub.2X.sup.30C,
--OCX.sup.30C.sub.3, --OCH.sub.2X.sup.300, --OCHX.sup.30C.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.3H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.30C is independently --F,
--Cl, --Br, or --I.
[0443] R.sup.31C is independently oxo,
halogen, --CX.sup.31C.sub.3, --CHX.sup.31C.sub.2,
--CH.sub.2X.sup.31C, --OCX.sup.31C.sub.3, --OCH.sub.2X.sup.31C,
--OCHX.sup.31C.sub.2, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2,
--NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH,
--NHOH, --N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.31C is independently --F, --Cl, --Br, or --I
[0444] L.sup.1 is --O--, --S--, substituted or unsubstituted
C.sub.1-C.sub.2 alkylene (e.g., --CH.sub.2--, --CH.sub.2CH.sub.2--,
--C(CH.sub.3)H--, or --CH(CH.sub.3)CH.sub.2--), or substituted or
unsubstituted 2 membered heteroalkylene (e.g., --CH.sub.2O--,
--OCH.sub.2--, --CH.sub.2S--, --SCH.sub.2--, --CH.sub.2NH--,
--NHCH.sub.2--, --CH(CH.sub.3)O--, --OCH(CH.sub.3)--, --C
H(CH.sub.3)S--, --SCH(CH.sub.3)--, --CH(CH.sub.3)NH--,
--NHCH(CH.sub.3)--, --CH.sub.2N(CH.sub.3)--, or
--N(CH.sub.3)CH.sub.2--). In embodiments, L.sup.1 is --O--, --S--,
or substituted or unsubstituted methylene. In embodiments, L.sup.1
is --SCH.sub.2--. In embodiments, L.sup.1 is --O--. In embodiments,
L.sup.1 is --S--. In embodiments, L.sup.1 is --CH(CH.sub.3)--.
[0445] In embodiments, L.sup.1 is a bond, --S(O).sub.2--,
--N(R.sup.3)--, --O--, --S--, --C(O)--, --C(O)N(R.sup.3)--,
--N(R.sup.3)C(O)--, --N(R.sup.3)C(O)NH--, substituted or
unsubstituted alkylene, or substituted or unsubstituted
heteroalkylene. In embodiments, L.sup.1 is a bond,
--C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--, substituted or
unsubstituted alkylene, or substituted or unsubstituted
heteroalkylene. In embodiments, L.sup.1 is --C(O)N(R.sup.3)--,
unsubstituted alkylene, or unsubstituted heteroalkylene. In
embodiments, L.sup.1 is --C(O)N(R.sup.3)--. In embodiments, L.sup.1
is unsubstituted alkylene. In embodiments, L.sup.1 is unsubstituted
heteroalkylene. In embodiments, L.sup.1 is --C(O)NH--.
[0446] In embodiments, L.sup.1 is
##STR00010##
In embodiments, L.sup.1 is
##STR00011##
In embodiments, L.sup.1 is in embodiments, L.sup.1 is
##STR00012##
In embodiments, L.sup.1 is
##STR00013##
In embodiments, L.sup.1 is
##STR00014##
In embodiments, L.sup.1 is
##STR00015##
[0447] In embodiments, L.sup.1 is independently --O--, --S--,
R.sup.32-substituted or unsubstituted C.sub.1-C.sub.2 alkylene
(e.g., C.sub.1 or C.sub.2) or R.sup.32-substituted or unsubstituted
2 membered heteroalkylene. In embodiments, L.sup.1 is
R.sup.32-substituted or unsubstituted alkylene (e.g.,
C.sub.1-C.sub.8 alkylene, C.sub.1-C.sub.6 alkylene, or
C.sub.1-C.sub.4 alkylene), R.sup.32-substituted or unsubstituted
heteroalkylene (e.g., 2 to 8 membered heteroalkylene, 2 to 6
membered heteroalkylene, or 2 to 4 membered heteroalkylene),
R.sup.32-substituted or unsubstituted cycloalkylene (e.g.,
C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.6 cycloalkylene, or
C.sub.5-C.sub.6 cycloalkylene), R.sup.32-substituted or
unsubstituted heterocycloalkylene (e.g., 3 to 8 membered
heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6
membered heterocycloalkylene), R.sup.32-substituted or
unsubstituted arylene (e.g., C.sub.6-C.sub.10 arylene, C.sub.10
arylene, or phenylene), or R.sup.32-substituted or unsubstituted
heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9
membered heteroarylene, or 5 to 6 membered heteroarylene). In
embodiments, L.sup.1 is independently --O--, --S--, unsubstituted
C.sub.1-C.sub.2 alkylene (e.g., C.sub.1 or C.sub.2) or
unsubstituted 2 membered heteroalkylene. In embodiments, L.sup.1 is
independently unsubstituted methylene. In embodiments, L.sup.1 is
independently unsubstituted ethylene. In embodiments, L.sup.1 is
substituted 2 membered heteroalkylene. In embodiments, L.sup.1 is
substituted 3 membered heteroalkylene. In embodiments, L.sup.1 is
substituted 4 membered heteroalkylene. In embodiments, L.sup.1 is
an unsubstituted 2 membered heteroalkylene. In embodiments, L.sup.1
is an unsubstituted 3 membered heteroalkylene. In embodiments,
L.sup.1 is an unsubstituted 4 membered heteroalkylene. In
embodiments, L.sup.1 is --CONHCH.sub.2--.
[0448] R.sup.32 is independently oxo,
halogen, --CX.sup.32.sub.3, --CHX.sup.32.sub.2, --CH.sub.2X.sup.32,
--OCX.sup.32.sub.3, --OCH.sub.2X.sup.32, --OCHX.sup.32.sub.2, --CN,
--OH, --NH.sub.2, --CO OH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.33-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.33-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.33-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.33-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.33-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.33-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.32 is independently oxo, halogen,
--CX.sup.32.sub.3, --CHX.sup.32.sub.2, --CH.sub.2X.sup.32,
--OCX.sup.32.sub.3, --OCH.sub.2X.sup.32, --OCHX.sup.32.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.32 is independently --F,
--Cl, --Br, or --I.
[0449] In embodiments, R.sup.32 is independently unsubstituted
methyl. In embodiments, R.sup.32 is independently unsubstituted
ethyl.
[0450] R.sup.33 is independently oxo,
halogen, --CX.sup.33.sub.3, --CHX.sup.33.sub.2, --CH.sub.2X.sup.33,
--OCX.sup.33.sub.3, --OCH.sub.2X.sup.33, --OCHX.sup.33.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.34-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.34-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.34-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.34-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.34-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.34-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.33 is independently oxo, halogen,
--CX.sup.33.sub.3, --CHX.sup.33.sub.2, --CH.sub.2X.sup.33,
--OCX.sup.33.sub.3, --OCH.sub.2X.sup.33, --OCHX.sup.33.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.33 is independently --F,
--Cl, --Br, or --I.
[0451] In embodiments, R.sup.33 is independently unsubstituted
methyl. In embodiments, R.sup.33 is independently unsubstituted
ethyl.
[0452] R.sup.34 is independently oxo,
halogen, --CX.sup.34.sub.3, --CHX.sup.34.sub.2, --CH.sub.2X.sup.34,
--OCX.sup.34.sub.3, --OCH.sub.2X.sup.34, --OCHX.sup.34.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.34 is independently --F,
--Cl, --Br, or --I.
[0453] In embodiments, R.sup.34 is independently unsubstituted
methyl. In embodiments, R.sup.34 is independently unsubstituted
ethyl.
[0454] In embodiments, L.sup.2 is --O--, --S--, substituted or
unsubstituted C.sub.1-C.sub.2 alkylene (e.g., --CH.sub.2--,
--CH.sub.2CH.sub.2--, --C(CH.sub.3)H--, or
--CH(CH.sub.3)CH.sub.2--), or substituted or unsubstituted 2
membered heteroalkylene (e.g., --CH.sub.2O--, --OCH.sub.2--,
--CH.sub.2S--, --SCH.sub.2--, --CH.sub.2NH--, --NHCH.sub.2--,
--CH(CH.sub.3)O--, --OCH(CH.sub.3)--, --CH(CH.sub.3)S--,
--SCH(CH.sub.3)--, --CH(CH.sub.3)NH--, --NHCH(C H.sub.3)--,
--CH.sub.2N(CH.sub.3)--, or --N(CH.sub.3)CH.sub.2--). In
embodiments, L.sup.2 is --O--, --S--, or substituted or
unsubstituted methylene. In embodiments, L.sup.2 is --SCH.sub.2--.
In embodiments, L.sup.2 is --O--. In embodiments, L.sup.2 is --S--.
In embodiments, L.sup.2 is --CH(CH.sub.3)--. In embodiments,
L.sup.2 is a bond, --S(O).sub.2--, --N(R.sup.4)--, --O--, --S--,
--C(O)--, --C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--,
--N(R.sup.4)C(O)NH--, substituted or unsubstituted alkylene, or
substituted or unsubstituted heteroalkylene. In embodiments,
L.sup.2 is a bond, --N(R.sup.4)C(O)--, or substituted
heteroalkylene. In embodiments, L.sup.2 is a bond. In embodiments,
L.sup.2 is --N(R.sup.4)C(O)--. In embodiments, L.sup.2 is
substituted heteroalkylene.
[0455] In embodiments. L.sup.2 is a bond. In embodiments. L.sup.2
is --O--. In embodiments. L.sup.2 is
##STR00016##
In embodiments, L.sup.2 is
##STR00017##
In embodiments, L.sup.2 is
##STR00018##
In embodiments, L.sup.2 is
##STR00019##
In embodiments, L.sup.2 is
##STR00020##
In embodiments, L.sup.2 is
##STR00021##
In embodiments, L.sup.2 is
##STR00022##
In embodiments, L.sup.2 is
##STR00023##
[0456] In embodiments, L.sup.2 is a bond, --S(O).sub.2--, --NH--,
--O--, --S--, --C(O)--, --C(O)NH--, --NHC(O)--, --NHC(O)NH--,
substituted or unsubstituted alkylene, or substituted or
unsubstituted heteroalkylene. In embodiments, L.sup.2 is a bond,
--NHC(O)--, or substituted heteroalkylene. In embodiments, L.sup.2
is a bond. In embodiments, L.sup.2 is --NHC(O)--. In embodiments,
L.sup.2 is substituted heteroalkylene.
[0457] In embodiments, L.sup.2 is substituted 2 to 6 membered
heteroalkylene. In embodiments, L.sup.2 is substituted 3 to 6
membered heteroalkylene. In embodiments, L.sup.2 is substituted 3
to 5 membered heteroalkylene. In embodiments, L.sup.2 is
substituted 2 membered heteroalkylene. In embodiments, L.sup.2 is
substituted 3 membered heteroalkylene. In embodiments, L.sup.2 is
substituted 4 membered heteroalkylene. In embodiments, L.sup.2is
substituted 3 membered heteroalkylene. In embodiments, L.sup.2 is
substituted 6 membered heteroalkylene. In embodiments, L.sup.2 is
--NHC(O)CH.sub.2CH.sub.2--. In embodiments, L.sup.2 is
--NHC(O)CH.sub.2--. In embodiments, L.sup.2 is a bond.
[0458] In embodiments, L.sup.2 is independently --O--, --S--,
R.sup.35-substituted or unsubstituted C.sub.1-C.sub.2 alkylene
(e.g., C.sub.1 or C.sub.2) or R.sup.35-substituted or unsubstituted
2 membered heteroalkylene. In embodiments, L.sup.2 is
R.sup.35-substituted or unsubstituted alkylene (e.g.,
C.sub.1-C.sub.8 alkylene, C.sub.1-C.sub.6 alkylene, or
C.sub.1-C.sub.4 alkylene), R.sup.35-substituted or unsubstituted
heteroalkylene (e.g., 2 to 8 membered heteroalkylene, 2 to 6
membered heteroalkylene, or 2 to 4 membered heteroalkylene),
R.sup.35-substituted or unsubstituted cycloalkylene (e.g.,
C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.6 cycloalkylene, or
C.sub.5-C.sub.6 cycloalkylene), R.sup.35-substituted or
unsubstituted heterocycloalkylene (e.g., 3 to 8 membered
heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6
membered heterocycloalkylene), R.sup.35-substituted or
unsubstituted arylene (e.g., C.sub.6-C.sub.10 arylene, C.sub.10
arylene, or phenylene), or R.sup.35-substituted or unsubstituted
heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9
membered heteroarylene, or 5 to 6 membered heteroarylene). In
embodiments, L.sup.2 is independently --O--, --S--, unsubstituted
C.sub.1-C.sub.2 alkylene (e.g., C.sub.1 or C.sub.2) or
unsubstituted 2 membered heteroalkylene. In embodiments, L.sup.2 is
independently unsubstituted methylene. In embodiments, L.sup.2 is
independently unsubstituted ethylene.
[0459] R.sup.35 is independently oxo,
halogen, --CX.sup.35.sub.3, --CHX.sup.35.sub.2, --CH.sub.2X.sup.35,
--OCX.sup.35.sub.3, --OCH.sub.2X.sup.35, --OCHX.sup.35.sub.2, --CN,
--OH, --NH.sub.2, --CO OH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.36-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.36-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 5 membered), R.sup.36-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.20-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.36-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.36-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.35 is independently oxo, halogen,
--CX.sup.35.sub.3, --CHX.sup.35.sub.2, --CH.sub.2X.sup.35,
--OCX.sup.35.sub.3, --OCH.sub.2X.sup.35, --OCHX.sup.35.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.35 is independently --F,
--Cl, --Br, or --I.
[0460] In embodiments, R.sup.3S is independently unsubstituted
methyl. In embodiments, R.sup.35 is independently unsubstituted
ethyl.
[0461] R.sup.36 is independently oxo,
halogen, --CX.sup.36.sub.3, --CHX.sup.36.sub.2, --CH.sub.2X.sup.36,
--OCX.sup.36.sub.3, --OCH.sub.2X.sup.36, --OCHX.sup.36.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.37-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
R.sup.37-substituted or unsubstituted heteroalkyl (e.g., 2 to 8
membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4
to 3 membered), R.sup.37-substituted or unsubstituted cycloalkyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.37-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.37-substituted or unsubstituted aryl (e.g., C.sub.6-C.sub.10
or phenyl), or R.sup.37-substituted or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In
embodiments, R.sup.3B is independently oxo, halogen,
--CX.sup.36.sub.3, --CHX.sup.36.sub.2, --CH.sub.2X.sup.36,
--OCX.sup.36.sub.3, --OCH.sub.2X.sup.36, --OCHX.sup.36.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 3 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.36 is independently --F,
--Cl, --Br, or --I.
[0462] In embodiments, R.sup.3B is independently unsubstituted
methyl. In embodiments, R.sup.36 is independently unsubstituted
ethyl.
[0463] R.sup.37 is independently oxo,
halogen, --CX.sup.37.sub.3, --CHX.sup.37.sub.2, --CH.sub.2X.sup.37,
--OCX.sup.37.sub.3, --OCH.sub.2X.sup.37, --OCHX.sup.37.sub.2, --CN,
--OH, --NH.sub.2, --COO H, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SC.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH,
--N.sub.3,unsubstituted alkyl (e.g., C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2),
unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered,
4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl
(e.g., C.sub.6-C.sub.10 or phenyl), or unsubstituted heteroaryl
(e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
X.sup.37 is independently --F, --Cl, --Br, or --I.
[0464] In embodiments, R.sup.37 is independently unsubstituted
methyl. In embodiments, R.sup.37 is independently unsubstituted
ethyl.
[0465] In embodiments, the compound as described herein, including
embodiments thereof, has the formula:
##STR00024##
wherein R.sup.23 is as described herein, including embodiments.
Ring A is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. The
symbol z23 is an integer from 0 to 3.
[0466] In embodiments, Ring A is (C.sub.3-C.sub.10) cycloalkyl, 3
to 10 membered heterocycloalkyl, (C.sub.6-C.sub.10) aryl, or 5 to
10 membered heteroaryl. In embodiments, Ring A is a heteroaryl. In
embodiments, Ring A is a 5 to 6 membered heteroaryl. In
embodiments, Ring A is a 5 membered heteroaryl.
[0467] In embodiments, Ring A is a (C.sub.3-C.sub.10) cycloalkyl, a
3 to 10 membered heterocycloalkyl, a (C.sub.6-C.sub.10) aryl, or a
5 to 10 membered heteroaryl. In embodiments, Ring A is a cycloalkyl
(e.g., C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl). In embodiments, Ring A is a
C.sub.3-C.sub.8 cycloalkyl. In embodiments, Ring A is a
C.sub.3-C.sub.6 cycloalkyl. In embodiments, Ring A is a
C.sub.5-C.sub.6 cycloalkyl. In embodiments, Ring A is a C.sub.6
cycloalkyl. In embodiments, Ring A is a C.sub.5 cycloalkyl. In
embodiments, Ring A is a (C.sub.6-C.sub.10) aryl. In embodiments,
Ring A is phenyl. In embodiments, Ring A is naphthyl. In
embodiments, Ring A is aziridinyl, oxiranyl, thiiranyl, azetidinyl,
oxetanyl, thietanyl, pyrrolidinyl, pyrrolyl, imidazolyl,
imidazolinyl, pyrazolinyl, tetrahydrofuranyl, thiolanyl,
piperidinyl, piperazinyl, pyranyl, morpholinyl, 1,4-dioxanyl,
tetrahydro-2H-pyranyl, thianyl, or dithianyl. In embodiments, Ring
A is a phenyl, thiofuranyl, imidazolyl, pyrazolyl, triazolyl,
tetrazolyl, furanyl, oxazolyl, isooxazolyl, oxadiazolyl,
oxatriazolyl, thienyl, thiazolyl, isothiazolyl, pyridinyl,
pyrazinyl, pyrimidinyl, pyridazinyl, or triazinyl (e.g.,
1,3,5-triazinyl, 1,2,3-triazinyl, or 1,2,4-triazinyl). In
embodiments, Ring A is indolyl, benzimidazolyl, indazolyl,
benzotriazolyl, pynolopyrimidinyl, purinyl, indolizinyl,
pyrrolopyriazinyl, pyrrolopyrimidinyl, imidazopyridazinyl,
imidazopyridinyl, imidazopyrimidinyl, cinnolinyl, quinazolinyl,
quinoxalinyl, phthalazinyl, pyridopyrazinyl, pteridinyl,
pyrazolopyridinyl, quinolinyl, isoquinolinyl, naphthyridinyl, or
carbazolyl.
[0468] In embodiments, -(Ring A)-(R.sup.23).sub.z23 is:
##STR00025##
wherein R.sup.23 and z23 are as described herein including
embodiments.
[0469] In embodiments, -(ring A)-(R.sup.23).sub.z23 is:
##STR00026##
wherein R.sup.23 is as described herein, including embodiments.
[0470] In embodiments, R.sup.23 is independently halogen,
--CX.sup.23.sub.3, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --NR.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl.
[0471] In embodiments, R.sup.23 is independently halogen,
--CX.sup.23.sub.3, C(O)R.sup.100C, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl. In embodiments, R.sup.23 is
substituted or unsubstituted phenyl. In embodiments, R.sup.23 is
substituted phenyl. In embodiments, R.sup.23 is unsubstituted
phenyl.
[0472] In embodiments, R.sup.23 is independently halogen,
--CX.sup.23.sub.3, C(O)R.sup.100C, R.sup.24-substituted or
unsubstituted heterocycloalkyl, R.sup.24-substituted or
unsubstituted aryl, or R.sup.24-substituted or unsubstituted
heteroaryl. In embodiments, R.sup.23 is R.sup.24-substituted or
unsubstituted phenyl. In embodiments, R.sup.23 is
R.sup.24-substituted phenyl.
[0473] In embodiments, R.sup.23 is R.sup.24-substituted or
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6 cycloalkyl). In
embodiments, R.sup.23 is R.sup.24-substituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl). In embodiments, R.sup.23 is an
unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6 cycloalkyl).
[0474] In embodiments, R.sup.23 is R.sup.24-substituted or
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl). In embodiments, R.sup.23 is
R.sup.24-substituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl). In embodiments, R.sup.23 is an
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered
heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6
membered heterocycloalkyl).
[0475] In embodiments, R.sup.23 is R.sup.24-substituted or
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R.sup.23 is R.sup.24-substituted aryl
(e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In
embodiments, R.sup.23 is an unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R.sup.23 is an unsubstituted phenyl. In embodiments, R.sup.23 is a
R.sup.24-substituted phenyl.
[0476] In embodiments, R.sup.23 is R.sup.20-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R.sup.23 is R.sup.24-substituted heteroaryl (e.g., 5
to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R.sup.23 is an unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments,
R.sup.23 is R.sup.20-substituted imidazolyl, R.sup.24-substituted
pyrrolyl, R.sup.24-substituted pyrazolyl, R.sup.24-substituted
triazolyl, R.sup.24-substituted tetrazolyl, R.sup.24-substituted
furanyl, R.sup.24-substituted oxazolyl, R.sup.24-substituted
isooxazolyl, R.sup.24-substituted oxadiazolyl, R.sup.24-substituted
oxatriazolyl, R.sup.24-substituted thienyl, R.sup.24-substituted
thiazolyl, R.sup.24-substituted isothiazolyl, R.sup.24-substituted
pyridinyl, R.sup.24-substituted pyrazinyl, R.sup.24-substituted
pyrimidinyl, R.sup.24-substituted pyridazinyl, R.sup.24-substituted
triazinyl (e.g., R.sup.24-substituted 1,3,5-triazinyl,
R.sup.24-substituted 1,2,3-triazinyl, or R.sup.24-substituted
1,2,4-triazinyl). In embodiments, R.sup.23 is an unsubstituted
imidazolyl, an unsubstituted pyrrolyl, an unsubstituted pyrazolyl,
an unsubstituted triazolyl, an unsubstituted tetrazolyl, an
unsubstituted furanyl, an unsubstituted oxazolyl, an unsubstituted
isooxazolyl, an unsubstituted oxadiazolyl, an unsubstituted
oxatriazolyl, an unsubstituted thienyl, an unsubstituted thiazolyl,
an unsubstituted isothiazolyl, an unsubstituted pyridinyl, an
unsubstituted pyrazinyl, an unsubstituted pyrimidinyl, an
unsubstituted pyridazinyl, an unsubstituted triazinyl (e.g., an
unsubstituted 1,3,5-triazinyl, an unsubstituted 1,2,3-triazinyl, or
an unsubstituted 1,2,4-triazinyl). In embodiments, R.sup.23 is an
unsubstituted thiofuranyl. In embodiments, R.sup.23 is
unsubstituted thienyl.
[0477] In embodiments, R.sup.24 is --Br. In embodiments, R.sup.24
is --Cl. In embodiments, R.sup.24 is --I. In embodiments, R.sup.24
is --F. In embodiments, R.sup.24 is --OCH.sub.3. In embodiments,
R.sup.24 is --OCH.sub.2CH.sub.3. In embodiments, R.sup.24 is
substituted or unsubstituted 2 to 3 membered heteroalkyl. In
embodiments, R.sup.24 is substituted or unsubstituted 2 to 4
membered heteroalkyl. In embodiments, R.sup.24 is substituted or
unsubstituted 2 to 6 membered heteroalkyl. In embodiments, R.sup.24
is --CF.sub.3. In embodiments, R.sup.24 is --CHF.sub.2. In
embodiments, R.sup.24 is --CH.sub.2F. In embodiments, R.sup.24 is
--CCl.sub.3. In embodiments, R.sup.24 is --CHCl.sub.2. In
embodiments, R.sup.24 is --CH.sub.2Cl. In embodiments, R.sup.24 is
--CBr.sub.3. In embodiments, R.sup.24 is --CHBr.sub.2. In
embodiments, R.sup.24 is --CH.sub.2Br. In embodiments, R.sup.24 is
--CI.sub.3. In embodiments, R.sup.24 is --CHI.sub.2. In
embodiments, R.sup.24 is --CH.sub.2I. In embodiments, R.sup.24 is
--OCF.sub.3. In embodiments, R.sup.24 is --OCHF.sub.2. In
embodiments, R.sup.24 is --OCH.sub.2F. In embodiments, R.sup.24 is
--OCCI.sub.3. In embodiments, R.sup.24 is --OCHCl.sub.2. In
embodiments, R.sup.24 is --OCH.sub.2Cl. In embodiments, R.sup.24 is
--OCBr.sub.3. In embodiments, R.sup.24 is --OCHBr.sub.2. In
embodiments, R.sup.24 is --OCH.sub.2Br. In embodiments, R.sup.24 is
--OCI.sub.3. In embodiments, R.sup.24 is --OCHI.sub.2. In
embodiments, R.sup.24 is --OCH.sub.2I.
[0478] In embodiments, R.sup.24 is substituted or unsubstituted
C.sub.1-C.sub.6 alkyl. In embodiments, R.sup.24 is substituted or
unsubstituted 2 to 6 membered heteroalkyl. In embodiments, R.sup.24
is substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl. In
embodiments, R.sup.24 is substituted or unsubstituted 3 to 8
membered heterocycloalkyl. In embodiments, R.sup.24 is substituted
or unsubstituted phenyl. In embodiments, R.sup.24 is substituted or
unsubstituted 5 to 6 membered heteroaryl. In embodiments, R.sup.24
is independently halogen. In embodiments, R.sup.24 is independently
--CX.sup.24.sub.3. In embodiments, R.sup.24 is independently
--CHX.sup.24.sub.2. In embodiments, R.sup.24 is independently
--CH.sub.2X.sup.24. In embodiments, R.sup.24 is independently
--OCX.sup.24.sub.3. In embodiments, R.sup.24 is independently
--OCH.sub.2X.sup.24. In embodiments, R.sup.24 is independently
--OCHX.sup.24.sub.2. In embodiments, R.sup.24 is independently
--OCH.sub.2X.sup.24. In embodiments, R.sup.24 is independently
--CN. In embodiments, R.sup.24 is independently --CH.sub.3. In
embodiments, R.sup.24 is independently --OCH.sub.3.
[0479] In embodiments, R.sup.24 is substituted or unsubstituted
alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R.sup.24 is substituted
alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R.sup.24 is an
unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6
alkyl, or C.sub.1-C.sub.4 alkyl).
[0480] In embodiments, R.sup.24 is substituted or unsubstituted
methyl. In embodiments, R.sup.24 is substituted or unsubstituted
C.sub.2 alkyl. In embodiments, R.sup.24 is substituted or
unsubstituted C.sub.3 alkyl. In embodiments, R.sup.24 is
substituted or unsubstituted C.sub.4 alkyl. In embodiments,
R.sup.24 is substituted or unsubstituted C.sub.5 alkyl. In
embodiments, R.sup.24 is substituted or unsubstituted C.sub.6
alkyl. In embodiments, R.sup.24 is substituted or unsubstituted
C.sub.7 alkyl. In embodiments, R.sup.24 is substituted or
unsubstituted C.sub.8 alkyl. In embodiments, R.sup.24 is
substituted methyl. In embodiments, R.sup.24 is substituted C.sub.2
alkyl. In embodiments, R.sup.24 is substituted C.sub.3 alkyl. In
embodiments, R.sup.24 is substituted C.sub.4 alkyl. In embodiments,
R.sup.24 is substituted C.sub.5 alkyl. In embodiments, R.sup.24 is
substituted C.sub.6 alkyl. In embodiments, R.sup.24 is substituted
C.sub.7 alkyl. In embodiments, R.sup.24 is substituted C.sub.8
alkyl. In embodiments, R.sup.24 is an unsubstituted methyl. In
embodiments, R.sup.24 is an unsubstituted C.sub.2 alkyl. In
embodiments, R.sup.24 is an unsubstituted C.sub.3 alkyl. In
embodiments, R.sup.24 is an unsubstituted C.sub.4 alkyl. In
embodiments, R.sup.24 is an unsubstituted C.sub.5 alkyl. In
embodiments, R.sup.24 is an unsubstituted Q alkyl. In embodiments,
R.sup.24 is an unsubstituted C.sub.7 alkyl. In embodiments,
R.sup.24 is an unsubstituted C.sub.8 alkyl.
[0481] R.sup.100A, R.sup.100B, R.sup.100C, and R.sup.100D are
independently hydrogen, --CX.sub.3, --CN, --COOH, --CONH.sub.2,
--CHX2, --CH2.times., substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl.
[0482] In embodiments, R.sup.100A is independently hydrogen,
--CX.sup.100A.sub.3, --CHX.sup.100A.sub.2, --CH.sub.2X.sup.100A,
--CN, --COOH, --CONH.sub.2, R.sup.101A-substituted or unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.4, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.101A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered or 4 to 5 membered),
R.sup.101A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.101A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.101A-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.101A-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.100A is independently
hydrogen, --CX.sup.100A.sub.3, --CHX.sup.100A.sub.2,
--CH.sub.2X.sup.100A, --CN, --COOH, --CONH.sub.2, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.100A is independently --F, --Cl, --Br,
or --I.
[0483] In embodiments, R.sup.100A is independently hydrogen, in
embodiments, R.sup.100A is independently unsubstituted methyl. In
embodiments, R.sup.100A is independently unsubstituted ethyl.
[0484] In embodiments, R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.101A-substituted or unsubstituted heterocycloalkyl (e.g., 3
to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered) or R.sup.101A-substituted or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered) or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.100A and R.sup.100B
substituents bonded to the same nitrogen atom may optionally be
joined to form a R.sup.101A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.100A and R.sup.100B substituents bonded to the same nitrogen
atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered).
[0485] R.sup.101A is independently oxo,
halogen, --CX.sup.101A.sub.3, --CHX.sup.101A.sub.2,
--CH.sub.2X.sup.101A, --OCX.sup.101A.sub.3, --OCH.sub.2X.sup.101A,
--OCHX.sup.101A.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.102A-substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.102A-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.102A-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.102A-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.102A-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.102A-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.101A is independently
oxo, halogen, --CX.sup.101A.sub.3, --CHX.sup.101A.sub.2,
--CH.sub.2X.sup.101A, --OCX.sup.101A.sub.3, --OCH.sub.2X.sup.101A,
--OCHX.sup.101A.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.101A is independently --F, --Cl, --Br,
or --I.
[0486] In embodiments, R.sup.101A is independently unsubstituted
methyl. In embodiments, R.sup.101A is independently unsubstituted
ethyl.
[0487] R.sup.102A is independently oxo,
halogen, --CX.sup.102A.sub.3, --CHX.sup.102A.sub.2,
--CH.sub.2X.sup.102A, --OCX.sup.102A.sub.3, --OCH.sub.2X.sup.102A,
--OCHX.sup.102A.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.102A is independently --F, --Cl, --Br,
or --I.
[0488] In embodiments, R.sup.100B is independently hydrogen,
--CX.sup.100B.sub.3, --CHX.sup.100B.sub.2, --CH.sub.2X.sup.100B,
--CN, --COOH, --CONH.sub.2, R.sup.101B-substituted or unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.101B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.101B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.101B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.101B-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.101B-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.100B is independently
hydrogen, --CX.sup.100B.sub.3, --CHX.sup.100B.sub.2,
--CH.sub.2X.sup.100B, --CN, --COOH, --CONH.sub.2, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.100B is independently --F, --Cl, --Br,
or --I.
[0489] In embodiments, R.sup.100B is independently hydrogen. In
embodiments, R.sup.100B is independently unsubstituted methyl. In
embodiments, R.sup.100B is independently unsubstituted ethyl.
[0490] In embodiments, R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form a
R.sup.101B-substituted or unsubstituted heterocycloalkyl (e.g., 3
to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered,
or 5 to 6 membered) or R.sup.101B-substituted or unsubstituted
heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered). In embodiments, R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form
an unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered) or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.100A and R.sup.100B
substituents bonded to the same nitrogen atom may optionally be
joined to form a R.sup.101B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered). In embodiments,
R.sup.100A and R.sup.100B substituents bonded to the same nitrogen
atom may optionally be joined to form an unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered).
[0491] R.sup.101B is independently oxo,
halogen, --CX.sup.101B.sub.3, --CHX.sup.101B.sub.2,
--CH.sub.2X.sup.101B, --OCX.sup.101B.sub.3, --OCH.sub.2X.sup.101B,
--OCHX.sup.101B.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.102B-substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.102B-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.102B-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.6, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.102B-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.102B-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.102B-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.101B is independently
oxo, halogen, --CX.sup.101B, --CHX.sup.101B.sub.2,
--CH.sub.2X.sup.101B, --OCX.sup.101B.sub.3, --OCH.sub.2X.sup.101B,
--OCHX.sup.101B.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.101B is independently --F, --Cl, --Br,
or --I.
[0492] In embodiments, R.sup.101B is independently unsubstituted
methyl. In embodiments, R.sup.101B is independently unsubstituted
ethyl.
[0493] R.sup.102B is independently oxo, halogen,
--CX.sup.102B.sub.3, --CHX.sup.102B.sub.2, --CH.sub.2X.sup.102B,
--OCX.sup.102B.sub.3, --OCH.sub.2X.sup.102B, --OCHX.sup.102B.sub.2,
--CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2, --NO.sub.2, --SH,
--SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2,
--NHSO.sub.2H, --NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.4, or C.sub.1-C.sub.2), unsubstituted heteroalkyl
(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.1-C.sub.4, or
C.sub.5-C.sub.6), unsubstituted heterocycloalkyl (e.g., 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), unsubstituted aryl (e.g., C.sub.6-C.sub.10 or
phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to
9 membered, or 5 to 6 membered). X.sup.102B is independently --F,
--Cl, --Br, or --I.
[0494] In embodiments, R.sup.100C is independently
hydrogen, --CX.sup.100C.sub.3, --CHX.sup.100C.sub.2,
--CH.sub.2X.sup.100C, --CN, --COOH, --CONH.sub.2,
R.sup.101C-substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.101C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.101C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.101C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.101C-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.101C-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.100C is independently
hydrogen, --CX.sup.100C.sub.3, --CHX.sup.100C.sub.2,
--CH.sub.2X.sup.100C, --CN, --COOH, --CONH.sub.2, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.100C is independently --F, --Cl, --Br,
or --I.
[0495] In embodiments, R.sup.100C is independently hydrogen. In
embodiments, R.sup.100C is independently unsubstituted methyl. In
embodiments, R.sup.100C is independently unsubstituted ethyl.
[0496] R.sup.101C is independently oxo,
halogen, --CX.sup.101C.sub.3, --CHX.sup.101C.sub.2,
--CH.sub.2X.sup.101C, --OCX.sup.101C.sub.3, --OCH.sub.2X.sup.101C,
--OCHX.sup.101C.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.102C-substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.102C-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.102C-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.102C-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.102C-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.102C-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.101C is independently
oxo, halogen, --CX.sup.101C.sub.3, --CHX.sup.101C.sub.2,
--CH.sub.2X.sup.101C, --OCX.sup.101C.sub.3, --OCH.sub.2X.sup.101C,
--OCHX.sup.101C.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.101C is independently --F, --Cl, --Br,
or --I.
[0497] In embodiments, R.sup.101C is independently unsubstituted
methyl. In embodiments, R.sup.101C is independently unsubstituted
ethyl.
[0498] R.sup.102C is independently oxo,
halogen, --CX.sup.102C.sub.3, --CHX.sup.102C.sub.2,
--CH.sub.2X.sup.102C, --OCX.sup.102C.sub.3, --OCH.sub.2X.sup.102C,
--OCHX.sup.102C.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.102C is independently --F, --Cl, --Br,
or --I.
[0499] In embodiments, R.sup.100D is independently hydrogen,
--CX.sup.100D.sub.3, --CHX.sup.100D.sub.2, --CH.sub.2X.sup.100D,
--CN, --COOH, --CONH.sub.2, R.sup.101D-substituted or unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.101D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.101D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.101D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.101D-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.101D-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.100D is independently
hydrogen, --CX.sup.100D.sub.3, --CHX.sup.100D.sub.2,
--CH.sub.2X.sup.100D, --CN, --COOH, --CONH.sub.2, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.100D is independently --F, --Cl, --Br,
or --I.
[0500] In embodiments, R.sup.100D is independently hydrogen. In
embodiments, R.sup.100D is independently unsubstituted methyl. In
embodiments, R.sup.100D is independently unsubstituted ethyl.
[0501] R.sup.101D is independently oxo,
halogen, --CX.sup.101D.sub.3, --CHX.sup.101D.sub.2,
--CH.sub.2X.sup.101D, --OCX.sup.101D.sub.3, --OCH.sub.2X.sup.101D,
--OCHX.sup.101D.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3,
R.sup.102D-substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), R.sup.102D-substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6
membered, 2 to 3 membered, or 4 to 5 membered),
R.sup.102D-substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), R.sup.102D-substituted or unsubstituted
heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6
membered, 4 to 5 membered, or 5 to 6 membered),
R.sup.102D-substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 or phenyl), or R.sup.102D-substituted or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). In embodiments, R.sup.101D is independently
oxo, halogen, --CX.sup.101D.sub.3, --CHX.sup.101D.sub.2,
--CH.sub.2X.sup.101D, --OCX.sup.101D.sub.3, --OCH.sub.2X.sup.101D,
--OCHX.sup.101D.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.101D is independently --F, --Cl, --Br,
or --I.
[0502] In embodiments, R.sup.101D is independently unsubstituted
methyl. In embodiments, R.sup.101D is independently unsubstituted
ethyl.
[0503] R.sup.102D is independently oxo,
halogen, --CX.sup.102D.sub.3, --CHX.sup.102D.sub.2,
--CH.sub.2X.sup.102D, --OCX.sup.102D.sub.3, --OCH.sub.2X.sup.102D,
--OCHX.sup.102D.sub.2, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC.dbd.(O)NHNH.sub.2, --NHC.dbd.(O)NH.sub.2, --NHSO.sub.2H,
--NHC.dbd.(O)H, --NHC(O)--OH, --NHOH, --N.sub.3, unsubstituted
alkyl (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.4, or
C.sub.1-C.sub.2), unsubstituted heteroalkyl (e.g., 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5
membered), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
unsubstituted aryl (e.g., C.sub.6-C.sub.10 or phenyl), or
unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered,
or 5 to 6 membered). X.sup.102D is independently --F, --Cl, --Br,
or --I.
[0504] In embodiments, the compound has the formula:
##STR00027##
wherein R.sup.6, R.sup.5, L.sup.1, R.sup.1, and z1 are as described
herein, including embodiments.
[0505] In embodiments, the compound has the formula:
##STR00028##
wherein R.sup.6, R.sup.5, L.sup.1, R.sup.1, and z1 are as described
herein, including embodiments.
[0506] In embodiments, the compound has the formula:
##STR00029##
wherein L.sup.1, L.sup.2, Ring A, R.sup.23, and z23 are as
described herein, including embodiments. R.sup.1.1 and R.sup.1.3
are each R.sup.1 at a fixed position on the attached ring.
R.sup.1.1 and R.sup.1.3 may be any substituent of R.sup.1 described
herein, including in any aspect, embodiment, example, figure, or
claim.
[0507] In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4
alkyl). In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently substituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently an unsubstituted
alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R.sup.1.1 and R.sup.1.3 are
each independently substituted or unsubstituted methyl. In
embodiments, R.sup.1.1 and R.sup.1.3 are each independently
substituted or unsubstituted C.sub.2 alkyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently substituted or
unsubstituted C.sub.3 alkyl. In embodiments, R.sup.1.1 and
R.sup.1.3 are each independently substituted or unsubstituted
C.sub.4 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently substituted or unsubstituted C.sub.5 alkyl. In
embodiments, R.sup.1.1 and R.sup.1.3 are each independently
substituted or unsubstituted C.sub.6 alkyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently substituted or
unsubstituted C.sub.7 alkyl. In embodiments, R.sup.1.1 and
R.sup.1.3 are each independently substituted or unsubstituted
C.sub.8 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently substituted methyl. In embodiments, R.sup.1.1 and
R.sup.1.3 are each independently substituted C.sub.2 alkyl. In
embodiments, R.sup.1.1 and R.sup.1.3 are each independently
substituted C.sub.3 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3
are each independently substituted C.sub.4 alkyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently substituted C.sub.5
alkyl. In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently substituted C.sub.6 alkyl. In embodiments, R.sup.1.1
and R.sup.1.3 are each independently substituted C.sub.7 alkyl. In
embodiments, R.sup.1.1 and R.sup.1.3 are each independently
substituted C.sub.8 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3
are each independently an unsubstituted methyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently an unsubstituted
C.sub.2 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently an unsubstituted C.sub.3 alkyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently an unsubstituted
C.sub.4 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently an unsubstituted C.sub.5 alkyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently an unsubstituted
C.sub.6 alkyl. In embodiments, R.sup.1.1 and R.sup.1.3 are each
independently an unsubstituted C.sub.7 alkyl. In embodiments,
R.sup.1.1 and R.sup.1.3 are each independently an unsubstituted
C.sub.8 alkyl.
[0508] In embodiments, the compound has the formula:
##STR00030##
wherein L.sup.1, Ring A, R.sup.23, R.sup.1, z1, and z23 are as
described herein, including embodiments.
[0509] In embodiments, the compound has the formula:
##STR00031##
wherein R.sup.1.1, R.sup.1.3, Ring A, R.sup.23, and z23 are as
described herein, including embodiments.
[0510] In embodiments, the compound has the formula:
##STR00032##
[0511] Formula III is also referred to herein as 113B7, PDZ1in, and
PDZ1i.
[0512] The compounds of Formula I, Formula II, Formula IIA, and
Formula III can be synthesized according to procedures described
herein, including in FIG. 6.
[0513] In some aspects of the invention, the compounds described
herein are provided as prodrugs. In this case, one or more than one
functional group, and/or one or more than one type of functional
group, is covalently attached to reactive group of the molecule,
e.g. to an OH, an NH, a carboxyl, etc. In some aspects, the
protecting group is removed by enzymatic and/or non-enzymatic
reactions, e.g. by hydrolysis, at or near the site of action (e.g.
at the site of a tumor). In other aspects, the protecting group
remains attached to the drug at the site of action, but does not
interfere with the activity of the drug, or at least does not
interfere to an extent that make the drug ineffective. In yet other
aspects, the protecting groups are selected so as to be gradually
removed non-enzymatically as the drug circulates, resulting in a
slow release over time of an active drug. Exemplary protecting
groups that may be used to make such prodrugs include but are not
limited to: amidomethyl esters (for carboxyl groups); acyloxymethyl
(for carboxyl groups); monomethoxytrityl (MMT) (for amino groups);
carbamoyl moieties (carbamate esters of amino acids);
pivaloyloxymethyl (POM, pivoxil, pivoxyl), benzoyl, acetyl,
trimethylsilyl, triethylsilyl, or methoxymethyl groups (for hydroxy
groups); etc. In some aspects, carboxyl groups of the molecule are
protected in this manner and the protecting group is removed in
vivo, e.g. the protecting group is a cleavable alkyl of aryl ester
at a C-terminal carboxylate.
[0514] In embodiments, n1 is 0. In embodiments, n1 is 1. In
embodiments, n1 is 2. In embodiments, n1 is 3. In embodiments, n1
is 4. In embodiments, m1 is 1. In embodiments, m1 is 2. In
embodiments, v1 is 1. In embodiments, v1 is 2.
[0515] In embodiments, z1 is 0. In embodiments, z1 is 1. In
embodiments, z1 is 2. In embodiments, z1 is 3. In embodiments, z1
is 4. In embodiments, z1 is 5. In embodiments, the symbol z1 is an
integer from 0 to 4. In embodiments, the symbol z1 is an integer
from 0 to 2. In embodiments, z23 is 0. In embodiments, z23 is 1. In
embodiments, z23 is 2. In embodiments, z23 is 3. In embodiments,
z23 is 4. In embodiments, z23 is 5. In embodiments, the symbol z23
is an integer from 0 to 4.
[0516] In embodiments, n2 is 0. In embodiments, n2 is 1. In
embodiments, n2 is 2. In embodiments, n2 is 3. In embodiments, n2
is 4. In embodiments, m2 is 1. In embodiments, m2 is 2. In
embodiments, v2 is 1. In embodiments, v2 is 2.
[0517] In embodiments, n23 is 0. In embodiments, n23 is 1. In
embodiments, n23 is 2. In embodiments, n23 is 3. In embodiments,
n23 is 4. In embodiments, m23 is 1. In embodiments, m23 is 2. In
embodiments, v23 is 1. In embodiments, v23 is 2.
[0518] In embodiments, the compound is a compound described herein,
for example a compound in Table 1. In embodiments the compound is
113B12, 113B11, 113B9, 113B7, 112H9, 113B8, B112G11, 112G4, 112G3,
112G2, 112G1, 112F12, 112F11, 112F10, 112F1, 112E12, 112E7, 112D11,
cmpd14, cmpd13, cmpd12, cmpd11, cmpd10, cmpd9, cmpd8, cmpd7, cmpd6,
cmpd5, cmpd4, cmpd3, cmpd2, cmpd1, or 30A9 as identified in Table
1.
[0519] In an aspect is provided a pharmaceutical compositions
including a compound as described herein, including embodiments
thereof, and a pharmaceutically acceptable salt.
III. Method of Treating Cancer
[0520] Also provided herein are methods of treating cancer in a
subject in need thereof.
[0521] In some aspects, the subject who is treated as described
herein has, in fact, not been diagnosed with cancer. Rather, the
subject is genetically prone to development of cancer (e.g. the
subject may be a woman with a harmful mutation in the PALB2, BRCA1
and/or BRCA2, tumor suppressor genes; c-Kit, APC, mutated p53, PTEN
deletion, Braf, are some common gene alterations that are currently
tested for predicting cancer risk; activation of oncogenes
including members of the Ras gene family, AEG-1 (MTDH), myc gene
family (C-myc, N-myc, L-myc); deregulated cell cycle genes such as
cyclin E1; etc. In these cases, the compounds of the invention are
administered prophylactically and prevent or slow and/or lessen the
extent of the cancer that develops and/or make the cancer more
treatable when is does occur.
[0522] In yet other aspects, the compounds disclosed herein are
used to treat subjects who are not diagnosed with cancer per se but
rather with a pre-cancerous condition. For example, individuals
with colon polyps that are benign, individuals with cervical
dysplasia, individuals at high risk for cancer based on familial
history, individuals exposed to a carcinogen potentially causing
cancer (through the skin, respiratory track, gastrointestinal
track, or other route of entry into the body), individuals who have
been positively diagnosed using a genetic test for cancer
(including containing potentially cancerous circulating tumor
cells, presence of positive cancer associated biomarkers (proteins
in plasma, miRNA in the blood) expressed in body fluids (blood,
urine, saliva, etc.), etc.
[0523] In embodiments, the subject who is treated as described
herein has been diagnosed with early stage cancer. In such cases,
the compounds described herein may be used alone or in combination
with other therapeutic modes to address the cancer and to prevent
its spread or progression. For example, patients with early stage
bladder or prostate cancer are treated to prevent invasion and
development of metastatic lesions. Patients who underwent surgery
to remove primary or metastatic tumor or have undergone treatment
with chemotherapy/radiotherapy/immunotherapy for treating cancers.
Potential applications of the current anti-invasive and
anti-metastatic molecules is to prevent secondary tumor and
metastasis development following conventional surgery or therapy
(radiation, chemotherapy, immunotherapy) for cancer. Treatment
would begin prior to surgery or other therapies (radiation,
chemotherapy, immunotherapy) and continue after surgery or
therapy.
[0524] In another aspect is provided a method of preventing or
treating cancer in a subject in need thereof, including
administering to the subject a therapeutically effective amount of
aa compound described herein, including embodiments thereof wherein
the therapeutically effective amount is sufficient to prevent or
treat the cancer.
[0525] In another aspect is provided a method of sensitizing cancer
cells to killing by radiation, including contacting the cancer
cells with an effective (e.g., therapeutically effective) amount of
a compound described herein, including embodiments thereof, wherein
the therapeutically effective amount is sufficient to sensitize the
cancer cells to killing by radiation.
[0526] In another aspect is provided a method of slowing or
preventing metastasis of cancer cells in a subject in need thereof
including administering to the subject an effective (e.g.,
therapeutically effective) amount of a compound described herein,
including embodiments thereof, wherein the therapeutically
effective amount is sufficient to slow or prevent the
metastasis.
[0527] In another aspect is provided a method of treating a
glioblastoma multiforme brain tumor in a subject in need thereof,
including performing surgery on the subject to debulk the
glioblastoma multi forme brain tumor; radiosensitizing remaining
tumor cells by administering to the subject a therapeutically
effective amount of at least one of the compounds of any of the
invention, wherein the therapeutically effective amount is
sufficient to sensitize the remaining tumor cells to killing by
radiation; and providing radiation therapy to the subject.
[0528] In an aspect is provided a method of treating a glioblastoma
multiforme brain tumor in a subject in need thereof. In
embodiments, the method includes performing surgery on the subject
to debulk the glioblastoma multiforme brain tumor. In embodiments,
the method includes radiosensitizing the remaining tumor cells by
administering to the subject a therapeutically effective amount of
at least one of the compounds as described herein, including
embodiments. In embodiments, the therapeutically effective amount
is sufficient to sensitize the remaining tumor cells to killing by
radiation. In embodiments, the method includes providing radiation
therapy to the subject.
[0529] It is contemplated that inhibiting MDA-9 activity through
binding of the MDA-9 PDZ1 domain by a PDZ1 domain binder is useful
for the treatment of cancer (e.g., cancers having increased MDA-9
expression). Thus, in an aspect is provided a method of inhibiting
MDA-9 protein activity, the method including contacting the MDA-9
protein with an effective amount of a PDZ1 domain binder, thereby
inhibiting MDA-9 activity.
[0530] As mentioned above, a PDZ1 domain binder as referred to
herein is a compound or composition as described herein, including
embodiments thereof, (e.g., small molecule, antibody, aptamer,
ligand) that selectively binds to a PDZ1 domain of an MDA-9
protein. In embodiments, a PDZ1 domain of an MDA-9 protein includes
the sequence of SEQ ID NO:1. In embodiments, a PDZ1 domain of an
MDA-9 protein is the sequence of SEQ ID NO: 1. In embodiments, the
PDZ1 domain binder binds the PDZ1 domain, thereby inhibiting PDZ1
domain function. In embodiments, the PDZ1 domain binder binds a
portion of the PDZ1 domain. In embodiments, the PDZ1 domain binder
binds a portion of the PDZ1 domain and an amino acid interface
region located between the PDZ1 domain and the PDZ2 domain. A
portion of a PDZ1 domain and/or an amino acid interface region
refers to less than all of the amino acids that make up the region.
For example, in embodiments, PDZ1 domain binder binds to less than
100% of the amino acids included in the PDZ1 domain. In
embodiments, PDZ1 domain binder binds to less than 90% of the amino
acids included in the PDZ1 domain. In embodiments, PDZ1 domain
binder binds to less than 80% of the amino acids included in the
PDZ1 domain. In embodiments, PDZ1 domain binder binds to less than
70% of the amino acids included in the PDZ1 domain. In embodiments,
PDZ1 domain binder binds to less than 60% of the amino acids
included in the PDZ1 domain. In embodiments, PDZ1 domain binder
binds to less than 50% of the amino acids included in the PDZ1
domain. In embodiments, PDZ1 domain binder binds to less than 40%
of the amino acids included in the PDZ1 domain. In embodiments,
PDZ1 domain binder binds to less than 30% of the amino acids
included in the PDZ1 domain. In embodiments, PDZ1 domain binder
binds to less than 20% of the amino acids included in the PDZ1
domain. In embodiments, PDZ1 domain binder binds to less than 15%
of the amino acids included in the PDZ1 domain. In embodiments,
PDZ1 domain binder binds to less than 10% of the amino acids
included in the PDZ1 domain. In embodiments, PDZ1 domain binder
binds to less than 9% of the amino acids included in the PDZ1
domain. In embodiments, PDZ1 domain binder binds to less than 8% of
the amino acids included in the PDZ1 domain. In embodiments, PDZ1
domain binder binds to less than 7% of the amino acids included in
the PDZ1 domain. In embodiments, PDZ1 domain binder binds to less
than 6% of the amino acids included in the PDZ1 domain. In
embodiments, PDZ1 domain binder binds to less than 5% of the amino
acids included in the PDZ1 domain. In embodiments, PDZ1 domain
binder binds to less than 4% of the amino acids included in the
PDZ1 domain. In embodiments, PDZ1 domain binder binds to less than
3% of the amino acids included in the PDZ1 domain. In embodiments,
PDZ1 domain binder binds to less than 2% of the amino acids
included in the PDZ1 domain. In embodiments, PDZ1 domain binder
binds to less than 1% of the amino acids included in the PDZ1
domain.
[0531] Similarly, in embodiments, PDZ1 domain binder binds to less
than 100% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 90% of the amino
acids included in the interface region. In embodiments, PDZ1 domain
binder binds to less than 80% of the amino acids included in the
interface region. In embodiments, PDZ1 domain binder binds to less
than 70% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 60% of the amino
acids included in the interface region. In embodiments, PDZ1 domain
binder binds to less than 50% of the amino acids included in the
interface region. In embodiments, PDZ1 domain binder binds to less
than 40% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 30% of the amino
acids included in the interface region. In embodiments, PDZ1 domain
binder binds to less than 20% of the amino acids included in the
interface region. In embodiments, PDZ1 domain binder binds to less
than 15% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 10% of the amino
acids included in the interface region. In embodiments, PDZ1 domain
binder binds to less than 9% of the amino acids included in the
interface region. In embodiments, PDZ1 domain binder binds to less
than 8% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 7% of the amino
acids included in the interface region. In embodiments, PDZ1 domain
binder binds to less than 6% of the amino acids included in the
interface region. In embodiments, PDZ1 domain binder binds to less
than 5% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 4% of the amino
acids included in the interface region. In embodiments, PDZ1 domain
binder binds to less than 3% of the amino acids included in the
interface region. In embodiments, PDZ1 domain binder binds to less
than 2% of the amino acids included in the interface region. In
embodiments, PDZ1 domain binder binds to less than 1% of the amino
acids included in the interface region.
[0532] In embodiments, the PDZ1 domain binder occludes about 5% of
the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
about 10% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes about 15% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes about 20% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes about 25% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes about 30%
of the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
about 35% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes about 40% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes about 45% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes about 50% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes about 55%
of the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
about 60% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes about 65% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes about 70% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes about 75% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes about 80%
of the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
about 85% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes about 90% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes about 91% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes about 92% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes about 93%
of the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
about 94% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes about 95% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes about 96% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes about 97% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes about 98%
of the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
about 99% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes 100% of the PDZ1 domain.
[0533] In embodiments, the PDZ1 domain binder occludes 5% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes 10% of
the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
15% of the PDZ1 domain. In embodiments, the PDZ1 domain binder
occludes 20% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes 25% of the PDZ1 domain. In embodiments, the PDZ1
domain binder occludes 30% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes 35% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes 40% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes 45% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes 50% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes 55% of
the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
60% of the PDZ1 domain. In embodiments, the PDZ1 domain binder
occludes 65% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes 70% of the PDZ1 domain. In embodiments, the PDZ1
domain binder occludes 75% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes 80% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes 85% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes 90% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes 91% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes 92% of
the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
93% of the PDZ1 domain. In embodiments, the PDZ1 domain binder
occludes 94% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes 95% of the PDZ1 domain. In embodiments, the PDZ1
domain binder occludes 96% of the PDZ1 domain. In embodiments, the
PDZ1 domain binder occludes 97% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes 98% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes 99% of the PDZ1
domain. In embodiments, the PDZ1 domain binder occludes 100% of the
PDZ1 domain.
[0534] In embodiments, the PDZ1 domain binder occludes at least 5%
of the PDZ1 domain. In embodiments, the PDZ1 domain binder occludes
at least 10% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 15% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 20% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes at least 25% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes at
least 30% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 35% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 40% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes at least 45% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes at
least 50% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 55% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 60% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes at least 65% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes at
least 70% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 75% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 80% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes at least 85% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes at
least 90% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 91% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 92% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes at least 93% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes at
least 94% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 95% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 96% of the PDZ1 domain. In
embodiments, the PDZ1 domain binder occludes at least 97% of the
PDZ1 domain. In embodiments, the PDZ1 domain binder occludes at
least 98% of the PDZ1 domain. In embodiments, the PDZ1 domain
binder occludes at least 99% of the PDZ1 domain. In embodiments,
the PDZ1 domain binder occludes at least 100% of the PDZ1
domain.
[0535] PDZ1 domain occlusion may be measured using methods known in
the art A Non-limiting example includes measuring the solvent
accessible surface area in the presence and absence of the PDZ1
domain binder.
[0536] In embodiments, the PDZ1 domain binder is a small molecule
(e.g., a compound described herein), an antibody, an aptamer, or a
ligand. In embodiments, the PDZ1 domain binder is a small molecule
(e.g., a compound as described herein). In embodiments, the PDZ1
domain binder is an antibody. In embodiments, the PDZ1 domain
binder is an aptamer. In embodiments, the PDZ1 domain binder is a
ligand.
[0537] In embodiments, the antibody may be linked (e.g.,
covalently, non-covalently) to a phosphorothioate nucleic acid,
(e.g., wherein the phosphorothioate nucleic acid facilitates
intracellular delivery of the antibody). In embodiments, the
aptamer may be linked (e.g., covalently, non-covalently) to a
phosphorothioate nucleic acid, (e.g., wherein the phosphorothioate
nucleic acid facilitates intracellular delivery of the aptamer). In
embodiments, the ligand may be linked (e.g., covalently,
non-covalently) to a phosphorothioate nucleic acid, (e.g., wherein
the phosphorothioate nucleic acid facilitates intracellular
delivery of the ligand). Alternative, in embodiments, the antibody
may be linked (e.g., covalently, non-covalently) to a ligand that
binds a cell-surface receptor, (e.g., thereby promoting
intracellular delivery of the antibody). In embodiments, the
aptamer may be linked (e.g., covalently, non-covalently) to a
ligand that binds a cell-surface receptor, (e.g., thereby promoting
intracellular delivery of the aptamer). In embodiments, the ligand
may be linked (e.g., covalently, non-covalently) to a ligand that
binds a cell-surface receptor, (e.g., thereby promoting
intracellular delivery of the ligand).
[0538] In embodiments, the small molecule is a compound as
described herein, including embodiments thereof.
[0539] In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 25
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 24
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 23
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 22
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 21
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 20
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 15
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 10
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 9
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 8
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 7
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 6
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 5
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 4
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 3
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 2
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 1
.mu.M. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 300
nM. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 100
nM. In embodiments, the PDZ1 domain binder (e.g., compound
described herein) binds a PDZ1 domain with a Kd of less than 50 nM.
In embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 20 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 10 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 5 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 1 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 0.5 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 0.1 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 0.05 nM. In
embodiments, the PDZ1 domain binder (e.g., compound described
herein) binds a PDZ1 domain with a Kd of less than 0.025 nM.
[0540] In embodiments, the ligand is a natural ligand of a PDZ1
domain. In embodiments, the ligand is a natural ligand of a PDZ1
domain or a portion thereof. The term "natural ligand" refers to a
naturally occurring ligand that binds a PDZ1 domain.
[0541] In an aspect is provided a method of treating cancer in a
subject in need thereof, the method including administering to the
subject an effective amount of a PDZ1 domain binder.
[0542] In embodiments, the PDZ1 domain binder is a compound
described herein, including in embodiments, an aspect, an example,
a figure, a claim, a scheme, or a table.
[0543] In embodiments, the method further includes administering to
the subject an anti-cancer agent. In embodiments, the anti-cancer
agent is radiation, 5-FU, sorafenib, taxol, temozolimide, or Mcl-1.
In embodiments, the anti-cancer agent is radiation. In embodiments,
the anti-cancer agent is 5-FU. In embodiments, the anti-cancer
agent is sorafenib. In embodiments, the anti-cancer agent is taxol.
In embodiments, the anti-cancer agent is temozolimide. In
embodiments, the anti-cancer agent is Mcl-1.
[0544] In embodiments, the method further includes administering to
the subject a second therapy. In embodiments, the second therapy is
radiation. In embodiments, the second therapy may be administered
before the PDZ1 domain binder (e.g., compound described herein). In
embodiments, the second therapy may be administered after the PDZ1
domain binder (e.g., compound described herein). In embodiments,
the second therapy may be administered concurrently with the PDZ1
domain binder (e.g., compound described herein). In embodiments,
the second therapy is radiation. In embodiments, the second therapy
is administration of a second therapeutic agent. In embodiments,
the second therapeutic agent is an anti-cancer agent. In
embodiments, the second therapeutic agent is a chemotherapeutic
agent. In embodiments, the second therapeutic agent is an agent for
treating glioblastoma. In embodiments, the second therapeutic agent
is an agent for treating breast cancer. In embodiments, the second
therapeutic agent is an agent for treating urothelial cancer. In
embodiments, the second therapeutic agent is an agent for treating
melanoma. In embodiments, the second therapeutic agent is an agent
for treating hepatocellular carcinoma. In embodiments, the second
therapeutic agent is an agent for treating colorectal cancer. In
embodiments, the second therapeutic agent is an agent for treating
neuroblastoma. In embodiments, the second therapeutic agent is an
agent for treating colon cancer. In embodiments, the second
therapeutic agent is an agent for treating gastric cancer. In
embodiments, the second therapeutic agent is an agent for treating
bladder cancer. In embodiments, the second therapeutic agent is an
agent for treating lung cancer. In embodiments, the second
therapeutic agent is an agent for treating pancreatic cancer. In
embodiments, the second therapeutic agent is an agent for treating
head and neck cancer.
[0545] It is further contemplated that the compositions provided
herein, including embodiments thereof, are useful for preventing or
reducing metastasis of cancer cells. The compositions provided
herein, including embodiments thereof, can accomplish prevention or
reduction of metastasis of cancer cells by inhibiting cancer cell
invasion and attachment of cancer cells and cancer-associated
angiogenesis. Cancer cell invasion refers to the ability of cancer
cells to become motile and infiltrate neighboring tissue or blood
vessels. Cancer cell attachment refers the ability of cancer cells
to adhere to blood vessel walls and extravasate, or to adhere to
neighboring tissue, thereby forming metastases.
[0546] Therefore, in an aspect is provided a method of preventing
metastasis of cancer cells in a subject in need thereof, the method
including administering to the subject an effective amount of a
PDZ1 domain binder.
[0547] In an aspect is provided a method of reducing metastasis of
cancer cells in a subject in need thereof, the method including
administering to the subject an effective amount of a PDZ1 domain
binder. In embodiments, the method of reducing is measured relative
to a control (e.g., the absence of the PDZ1 domain binder).
[0548] In an aspect is provided a method of inhibiting cancer
associated angiogenesis in a subject in need thereof, the method
including administering to the subject an effective amount of a
PDZ1 domain binder. In embodiments, the method of inhibiting is
measured relative to a control (e.g., the absence of the PDZ1
domain binder).
[0549] In embodiments, the cancer is associated with an increased
MDA-9 gene expression. In embodiments, the cancer cells are
associated with an increased MDA-9 gene expression. Detection of
increased MDA-9 gene expression may be determined, for example, by
comparing MDA-9 gene expression from a biological sample (e.g.,
tumor biopsy, blood) obtained from a patient against a control
sample. The control sample may be a biological sample (e.g.,
healthy tissue) taken from the same patient. Alternatively, the
control sample may be a biological sample (e.g., tissue, blood)
obtained from a cancer-free subject. In embodiments, an increased
MDA-9 gene expression level is at least 1.02, 1.03, 1.04, 1.05,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,
4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 150, 200, 250, or 300 times greater than the
expression level observed in the control sample.
[0550] Detection of increased MDA-9 gene expression may also be
determined by comparing the level of MDA-9 gene expression in a
biological sample (e.g., tumor biopsy, blood) obtained from a
patient against MDA-9 expression levels from biological samples
obtained from a population of patients. Population data may be
obtained from public genome-wide expression databases. In
embodiments, an increased MDA-9 gene expression level is equal to
or greater than the mean of the MDA-9 expression level of the
patient population. In embodiments, an increased MDA-9 gene
expression level is greater than the median of the MDA-9 expression
level of the patient population. In embodiments, an increased
expression level of MDA-9 may be a relative expression level of at
least 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300.
The relative expression level value (z) can be quantified by the
following equation:
z=(I.sub.n-Average I.sub.norm)/standard deviation.sub.norm,
wherein n refers to every sample in the dataset (including tumors)
and norm refers to normal samples only.
[0551] In embodiments, the cancer is melanoma, glioblastoma, head
and neck cancer, urothelial cancer, breast cancer, uveal melanoma,
gastric cancer, lung adenocarcinoma, hepatocellular carcinoma,
colorectal cancer, prostate cancer, pancreatic cancer, or
neuroblastoma. In embodiments, the cancer is melanoma. In
embodiments, the cancer is glioblastoma. In embodiments, the cancer
is head and neck cancer. In embodiments, the cancer is urothelial
cancer. In embodiments, the cancer is breast cancer. In
embodiments, the cancer is uveal melanoma. In embodiments, the
cancer is gastric cancer. In embodiments, the cancer is lung
adenocarcinoma. In embodiments, the cancer is hepatocellular
carcinoma. In embodiments, the cancer is colorectal cancer. In
embodiments, the cancer is prostate cancer. In embodiments, the
cancer is pancreatic cancer. In embodiments, the cancer is
neuroblastoma.
[0552] In embodiments, the cancer cells are melanoma, glioblastoma,
head and neck cancer, urothelial cancer, breast cancer, uveal
melanoma, gastric cancer, lung adenocarcinoma, hepatocellular
carcinoma, colorectal cancer, prostate cancer, pancreatic cancer,
or neuroblastoma cancer cells. In embodiments, the cancer cells are
melanoma cancer cells. In embodiments, the cancer cells are
glioblastoma cancer cells. In embodiments, the cancer cells are
head and neck cancer cells. In embodiments, the cancer cells are
urothelial cancer cells. In embodiments, the cancer cells are
breast cancer cells. In embodiments, the cancer cells are uveal
melanoma cancer cells. In embodiments, the cancer cells are gastric
cancer cells. In embodiments, the cancer cells are lung
adenocarcinoma cancer cells. In embodiments, the cancer cells are
hepatocellular carcinoma cancer cells. In embodiments, the cancer
cells are colorectal cancer cells. In embodiments, the cancer cells
are prostate cancer cells. In embodiments, the cancer cells are
pancreatic cancer cells. In embodiments, the cancer cells are
neuroblastoma cancer cells.
[0553] In embodiments, the compound (e.g., compound described
herein) or method includes reducing the level of activity of NF-kB.
In embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of c-Src. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of p38 MAPK. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of a c-Src/p38 MAPK
signaling pathway. In embodiments, the compound (e.g., compound
described herein) or method includes reducing the level of activity
of MMP2. In embodiments, the compound (e.g., compound described
herein) or method includes reducing the level of activity of FAK.
In embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of EphA2. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of EGFR. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of EGFRvIII. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of MMP9. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of IGF-1R. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of STAT3. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of IGFBP-2. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of RhoA. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of Cdc42. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of TGF.beta.1. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of Slug. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of Snail. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of Zeb1. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of N-cadherin. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of IL-8. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of cyclin D1. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of CDK4. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of PI3K. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of CTNNB1. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of integrin .beta.1.
In embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of Akt. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of HIF-1.alpha.. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of VEGF-A. In
embodiments, the compound (e.g., compound described herein) or
method includes reducing the level of activity of AEG-1.
[0554] In embodiments, the cancer is melanoma. In embodiments, the
cancer is gastric cancer. In embodiments, the cancer is bladder
cancer. In embodiments, the cancer is glioblastoma. In embodiments,
the cancer is lung cancer. In embodiments, the cancer is small cell
lung cancer. In embodiments, the cancer is hepatoma. In
embodiments, the cancer is head and neck cancer. In embodiments,
the cancer is breast cancer. In embodiments, the cancer is triple
negative breast cancer. In embodiments, the cancer is prostate
cancer. In embodiments, the cancer is hormone refractory prostate
cancer. In embodiments, the cancer is hormone sensitive prostate
cancer. In embodiments, the cancer is urothelial cancer. In
embodiments, the cancer is uveal melanoma. In embodiments, the
cancer is adenocarcinoma. In embodiments, the cancer is
hepatocellular carcinoma. In embodiments, the cancer is colorectal
cancer. In embodiments, the cancer is pancreatic cancer. In
embodiments, the cancer is neuroblastoma. In embodiments, the
cancer is colon cancer.
IV. Method of Tresting Additional Diseases
[0555] In yet other aspects, the invention provides methods of
treating human diseases other than cancer that are associated with
elevated expression of mda-9. Examples of such diseases include but
are not limited to: asthma, neurodegenerative diseases and
infectious diseases including viral infection (e.g., HIV, SARS,
HPV, influenza), virus shedding (HIV), bacterial colonization in
the human gastrointestinal tract, etc.
[0556] In an aspect is provided a method of treating an
inflammatory disease in a subject in need thereof the method
including administering to the subject an effective amount of a
PDZ1 domain binder (e.g., compound described herein).
[0557] In an aspect is provided a method of treating a
neurodegenerative disease in a subject in need thereof, the method
including administering to the subject an effective amount of a
PDZ1 domain binder (e.g., compound described herein).
[0558] In an aspect is provided a method of treating an infectious
disease in a subject in need thereof the method including
administering to the subject an effective amount of a PDZ1 domain
binder (e.g., compound described herein).
V. Formulations
[0559] The compounds described herein are generally delivered
(administered) as a pharmaceutical composition. Such pharmaceutical
compositions generally include at least one of the disclosed
compounds (e.g., in pure form, salt, or prodrugs), and more than
one (a plurality) of different compounds (e.g. 2 or more such as 2,
3, 4, 5, 6, 7, 8, 9, 10 or more) may be included in a single
formulation. Accordingly, the present invention encompasses such
formulations and compositions. The compositions generally include
one or more substantially purified compounds as described herein,
and a pharmacologically suitable (physiologically compatible)
carrier, which may be aqueous or oil-based. In some embodiments,
such compositions are prepared as liquid solutions or suspensions.
In other embodiments, the compositions are prepared in solid forms
such as tablets, pills, powders and the like. Solid forms suitable
for solution, dissolution or suspension in liquids prior to
administration are also contemplated (e.g. lyophilized forms of the
compounds), as are emulsified preparations. In some embodiments,
the liquid formulations are aqueous or oil-based suspensions or
solutions. In some embodiments, the active ingredients are mixed
with excipients which are pharmaceutically acceptable and
compatible with the active ingredients, e.g. pharmaceutically
acceptable salts. Suitable excipients include, for example, water,
saline, dextrose, glycerol, cyclodextrin, ethanol and the like, or
combinations thereof. In addition, the composition may contain
minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, preservatives, and the
like. If it is desired to administer an oral form of the
composition, various thickeners, flavorings, diluents, emulsifiers,
dispersing aids or binders and the like are added. The composition
of the present invention may contain any such additional
ingredients so as to provide the composition in a form suitable for
administration. The final amount of compound in the formulations
varies, but is generally from about 1-99%. Still other suitable
formulations for use in the present invention are found, for
example in Remington's Pharmaceutical Sciences, 22nd ed. (2012;
eds. Allen, Adqarem Desselle and Felton).
[0560] Some examples of materials which serve as pharmaceutically
acceptable carriers include, but are not limited to, ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins
(such as human serum albumin), buffer substances (such as Tween 80,
phosphates, glycine, soibic acid, or potassium sorbate), partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes (such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, or zinc
salts), colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, methyl cellulose,
hydroxypropyl methylcellulose, wool fat, sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil;
safflower oil; sesame oil; olive oil; corn oil and soybean oil;
glycols; such a propylene glycol or polyethylene glycol; esters
such as ethyl oleate and ethyl laurate; agar; buffering agents such
as magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water, isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
[0561] "Pharmaceutically acceptable salts" refers to the relatively
non-toxic, inorganic and organic acid addition salts, and base
addition salts, of compounds (e.g., compound described herein).
These: salts can be prepared in situ during the final isolation and
purification of the compounds. In particular, acid addition salts
can be prepared by separately reacting the purified compound in its
free base form with a suitable organic or inorganic acid and
isolating the salt thus formed. Exemplary acid addition salts
include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate,
stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,
citrate, maleate, fumarate, succinate, tartrate, naphthylate,
mesylate, glucoheptonate, lactiobionate, sulfamates, malonates,
salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates,
gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates,
ethanesulfonates, benzenesulfonates, p-toluenesulfonates,
cyclohexylsulfamates and laurylsulfonate salts, and the like. See,
for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm.
Sci., 66, 1-19 (1977) which is incorporated herein by reference.
Base addition salts can also be prepared by separately reacting the
purified compound in its acid form with a suitable organic or
inorganic base and isolating the salt thus formed. Base addition
salts include pharmaceutically acceptable metal and amine salts.
Suitable metal salts include the sodium, potassium, calcium,
barium, zinc, magnesium, and aluminum salts. The sodium and
potassium salts are preferred. Suitable inorganic base addition
salts are prepared from metal bases which include sodium hydride,
sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum
hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide
and the like. Suitable amine base addition salts are prepared from
amines which have sufficient basicity to form a stable salt, and
preferably include those amines which are frequently used in
medicinal chemistry because of their low toxicity and acceptability
for medical use, ammonia, ethylenediamine, N-methyl-glucamine,
lysine, arginine, ornithine, choline, N,N'-di benzyl
ethylenediamine, chloroprocaine, diethanolamine, procaine,
N-benzylphenethylamine, diethylamine, piperazine,
tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide,
triethylamine, dibenzylamine, ephenamine, dehydroabietylamine,
N-ethylpiperidine, benzylamine, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
ethyl amine, basic amino acids, e.g., lysine and arginine, and
dicyclohexylamine, and the like.
VI. Administration
[0562] In embodiments, the formulation is administered in vivo by
any suitable route including but not limited to: inoculation or
injection (e.g. intravenous, intraperitoneal, intramuscular,
subcutaneous, intra-aural, intraarticular, intramammary,
intracranial, and the like), topical application (e.g. on any
suitable skin or membrane surface), by absorption through
epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal,
rectal, gastrointestinal mucosa, etc.) and the like. In
embodiments, the compounds may be incorporated into implantable
delivery means, e.g. drug permeated wafers, etc. Timed (sustained,
extended, controlled) release formulations, e.g. in the form of
pills, tablets, capsules, etc. are also contemplated, including
various diffusion systems such as reservoir and matrix devices;
osmotic, ion exchange, floating, bio-adhesive, and depot systems,
etc. In embodiments, the mode of administration is intranasal,
orally or parenteral, by intravenous, intraperitoneal,
intramuscular, topical or subcutaneous routes, and usually is by
intravenous injection.
[0563] In addition, in embodiments, the compounds may be
administered in conjunction with other treatment modalities.
Examples of such additional treatments include but are not limited
to: administration of various anti-cancer agents, for example,
cytotoxic chemotherapy agents; radiation; hormone therapy; T-cell
therapy; various immune checkpoint inhibitors; various forms of
targeted therapy (e.g. small molecules such as tyrosine-kinase
inhibitors, folate receptor inhibitors, serine/threonine kinase
inhibitors monoclonal antibodies, etc.).
[0564] In some aspects, the compounds as described herein,
including embodiments, administered with or in conjunction with one
or more anti-invasive and/or anti-attachment therapies. In some
aspects, the compounds of the invention are administered with or in
conjunction with one or more anti-cancer agents. Such combinations
result in induction of apoptosis and/or toxic autophagy and death
of cancer cells. For example, in embodiments, the present drugs can
be combined with PDZ inhibitors, with radiation (e.g. for GBM) with
sorafenib (for liver cancer), with taxol (for breast cancer),
etc.
[0565] Administration of other therapies that are not specific for
cancer, together with the compounds disclosed herein, is also
contemplated, e.g. appetite stimulants, nutritional supplements;
pain medication, anti-depressants, etc.
[0566] Administering a compound, antibody, aptamer, or ligand, as
described herein, including embodiments, "with" or "in conjunction
with" another agent may refer to administering a single composition
that includes both agents, but more typically refers to
administering one or more compounds of the invention to the same
subject during a course of treatment with the other agent, e.g.
more or less consecutively, or on the same day but several hours
apart, or several days (or even weeks) apart. In embodiments, the
compound, antibody, aptamer, or ligand, as described herein,
including embodiments, and the additional treatment (e.g.,
anti-cancer agent) are administered consecutively. In embodiments,
compound, antibody, aptamer or ligand, as described herein,
including embodiments, additional treatment (e.g., anti-cancer
agent) are administered simultaneously. In embodiments, compound,
antibody, aptamer or ligand, as described herein, including
embodiments, are admixed with additional treatment (e.g.,
anti-cancer agent) prior to administration.
[0567] In embodiments, administration is carried out in a
coordinated manner at time intervals which are spaced apart by
minutes, hours, days or weeks, etc. and the use of the multiple
agents is thus integrated into a treatment protocol in which the
effects are at least additive (i.e., a combined additive amount),
and may be synergistic (i.e., a combined synergistic amount).
[0568] A "combined additive amount" as used herein refers to the
sum of a first amount of a first agent (e.g., compound as described
herein) and a second amount of a second agent (e.g., an anti-cancer
agent), that results in an additive effect (i.e. an effect equal to
the sum of the effects). Therefore, the terms "additive", "combined
additive amount", and "additive therapeutic effect" which are used
herein interchangeably, refer to a measured effect of compounds
administered in combination where the measured effect is equal to
the sum of the individual effects of each of the compounds
administered alone as a single agent.
[0569] A "combined synergistic amount" as used herein refers to the
sum of a first amount of first agent (e.g., compound as described
herein) and a second amount of a second agent (e.g., an anti-cancer
agent) that results in a synergistic effect (i.e. an effect greater
than an additive effect). Therefore, the terms "synergy",
"synergism", "synergistic", "combined synergistic amount", and
"synergistic therapeutic effect" which are used herein
interchangeably, refer to a measured effect of compounds
administered in combination where the measured effect is greater
than the sum of the individual effects of each of the compounds
administered alone as a single agent.
[0570] In embodiments, a combined synergistic amount may be about
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,
4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8.4.9, 5.0, 5.1, 5.2,
5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,
6.6, 6.7, 6.8, 6.9.7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,
7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
of the amount of the first amount (e.g., PDZ1 domain binder) when
used separately from the second amount (e.g., anti-cancer agent).
In embodiments, a combined synergistic amount may be about 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,
2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,
4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,
5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,
8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,
9.3, 9.4, 9.5, 9.6, 9.7, 9.8.9.9, 10.0, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the
amount of the second amount (e.g., anti-cancer agent) when used
separately from the first amount (e.g., PDZ1 domain binder).
[0571] Of particular interest is administration of the present
agents together with radiation therapy, which are shown herein,
results in a synergistic effect in that more cancer cells are
killed by the combination than would be predicted if the effect was
purely additive, when based on results obtained by administering
each agent alone.
[0572] In some embodiments, the compounds of the invention are used
to block and/or prevent metastatic seeding of cells in the
circulatory system. Such methods are carried out by pretreating the
patient with a compound (e.g., compound described herein), i.e.
treating the patient before another cancer treatment is begun, and
then performing at least one other cancer treatment. The second
("other") cancer treatment may be, for example, surgical removal of
the tumor, and may be performed in the presence of any of the
compounds described herein. Thereafter, the method may include
continuing to treat the patient for a suitable period of time after
removing the tumor.
[0573] In yet other embodiments, the compounds of the invention are
used to block and/or prevent metastatic seeding. In such methods, a
patient with early stage cancer is treated with a compound (e.g.,
compound described herein), thereby preventing invasion and
shedding of tumor cells into the bloodstream and preventing
secondary seeding (colonization) of tumor cells in a distant site
(metastasis).
[0574] Before exemplary embodiments of the present invention are
described in greater detail, it is to be understood that this
invention is not limited to particular embodiments described, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting.
[0575] Where a range of values is provided, it is understood that
each intervening value between the upper and lower limit of that
range (to a tenth of the unit of the lower limit) is included in
the range and encompassed within the invention, unless the context
or description clearly dictates otherwise. In addition, smaller
ranges between any two values in the range are encompassed, unless
the context or description clearly indicates otherwise.
[0576] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Representative illustrative methods and materials are herein
described; methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present invention.
[0577] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference, and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual dates of public availability and may need
to be independently confirmed.
[0578] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as support for the
recitation in the claims of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitations, such as "wherein [a
particular feature or element] is absent", or "except for [a
particular feature or element]", or "wherein [a particular feature
or element] is not present (included, etc.) . . . ".
[0579] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
EXAMPLES
[0580] The following examples illustrate certain specific
embodiments of the invention and are not meant to limit the scope
of the invention.
[0581] Embodiments herein are further illustrated by the following
examples and detailed protocols. However, the examples are merely
intended to illustrate embodiments and are not to be construed to
limit the scope herein. The contents of all references and
published patents and patent applications cited throughout this
application are hereby incorporated by reference.
Example 1. Inhibition of Radiation-Induced Glioblastoma Invasion by
Genetic and Pharmacological Targeting of MDA-9/Syntenin (SDCBP)
[0582] Glioblastoma multiforme (GBM) is an intractable tumor
despite therapeutic advances principally because of its invasive
properties. Radiation is a staple in modern therapeutic regimens,
although cells surviving radiation can become more aggressive and
invasive. Subtraction hybridization identified melanoma
differentiation associated gene-9 (MDA-9/Syntenin; Syndecan Binding
Protein (SDCBP)), as a differentially regulated gene associated
with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a
highly conserved double PDZ domain-containing scaffolding protein,
is robustly expressed in human-derived GBM cell lines and patient
samples, with expression increasing with tumor grade and
correlating with shorter survival times and poorer response to
radiotherapy. Knockdown (kd) of MDA-9/Syntenin sensitizes GBM cells
to radiation, significantly reducing post-radiation invasion gains.
Radiation induces Src and EGFRvIII signaling, which is abrogated
through MDA-9/Syntenin downregulation. A novel exemplary inhibitor
of MDA-9/Syntenin activity, PDZ1i (113B7), was identified through
Nuclear Magnetic resonance (NMR) guided Fragment-Based Drug Design
(FBDD). PDZ1i treatment inhibited MDA-9/Syntenin binding to
EGFRvIII, which was increased following radiation. Both genetic
(shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin
reduced invasion gains in GBM cells following radiation. Although
not affecting normal astrocyte survival when combined with
radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial
GBM signaling involving FAK and mutant EGFR, EGFRvIII, and
abrogated gains in secreted proteases, MMP2 and MMP9, following
radiation. In an in vivo glioma model, PDZ1i treatment resulted in
smaller, less invasive tumors and enhanced survival. When combined
with radiation, survival gains exceeded radiotherapy alone. Small
molecule MDA-9/Syntenin inhibitors, like PDZ1i, hold promise to
advance targeted therapy of aggressive cancers like GBM.
[0583] GBM invasion into normal brain compounded by an inability to
surgically eliminate complete tumor contribute to GBM lethality and
recurrence. The "gold standard" for GBM therapy is radiation.
Unfortunately, evading radiation toxicity leads to further
therapeutic resistance. SDCBP (MDA-9/Syntenin) expression is
elevated in patient-derived samples and GBM cell lines, which
correlates with decreased survival and poor response to radiation.
Genetic suppression of MDA-9 expression sensitizes GBM to radiation
by inhibiting radiation-induced invasion gains and signaling
changes. Using fragment-based drug-discovery combined with NMR a
novel small molecule MDA-9/Syntenin inhibitor, PDZ1i, was
identified. PDZ1i improved survival of brain tumor bearing mice,
which was enhanced further when used with radiation supporting the
potential of PDZ1i as a novel therapeutic for this deadly
disease.
[0584] Despite advances in surgical, pharmacological, and radiation
therapeutic approaches, GBM remains a particularly aggressive and
ultimately an invariably fatal tumor with a median survival less
than 15 months and 5-year survival at 5%. The current standard of
care includes maximal surgical resection followed by radiation and
temozolomide chemotherapy. However, inevitable recurrence occurs
near resection margins and within the high-dose radiation field,
implying that intrinsic invasiveness and radioresistance
contributes significantly to relapse. While highly effective in
inducing cytotoxicity in a majority of tumor cells, sub-lethal
radiation has repeatedly been shown to induce invasion and
migration in surviving tumor cells, enhancing the very property
that makes curative treatment so difficult. A contributing factor
to tumor relapse and recurrence is the ability of tumor cells to
escape from the primary tumor mass, which underscores the
importance of developing anti-invasive therapies that complement,
and ideally enhance, conventional therapeutic approaches.
Therefore, gaining a deeper understanding of the crucial molecular
signaling events and regulatory molecules will help identify
targets for anti-invasive and radiosensitizing approaches.
[0585] Melanoma differentiation associated gene-9 (mda-9), also
known as syntenin (Syndecan Binding Protein; SDCBP) has been
demonstrated in multiple cancer settings to be involved in invasion
and metastatic signaling. MDA-9/Syntenin serves critical roles in
signal transduction, as well as in cell-cell, and cell-matrix
adhesion. In addition to its well-described roles in melanoma
metastasis and tumor progression, MDA-9/Syntenin was shown to be
highly expressed and involved in breast, gastric, and urothelial
cell cancers. In recent work, we showed that MDA-9/Syntenin is an
important regulator of GBM invasion, angiogenesis, and tumor
progression, and that inhibiting the expression of MDA-9/Syntenin
can decrease GBM invasion, and enhance survival. Gene expression
analysis of the TCGA database revealed that patients whose tumors
express high levels of MDA-9/Syntenin have a poor prognosis and
reduced survival compared to low-expressing MDA-9/Syntenin tumors,
mda-9/syntenin expression correlates positively with astrocytoma
grade, as analyzed through tissue samples and gene expression
databases, and is most highly expressed in GBM. In both melanoma
and glioma, MDA-9/Syntenin is involved in NF-kB activation through
a c-Src/p38 MAPK signaling pathway. Inhibiting MDA-9/Syntenin
expression can lead to a reduction in NF-kB target gene expression
such as MMP2, a critical secreted metalloproteinase involved in GBM
invasion.
[0586] A vital characteristic of MDA-9/syntenin is the inclusion of
two tandem PDZ domains, so named for their discovery in
PSD95/SAP90, DLGA, and ZO-1. PDZ domains are common to a number of
scaffolding proteins, critical for facilitating protein-protein
interactions throughout various regions of the cell. MDA-9/Syntenin
utilizes these motifs to successfully facilitate the interaction of
c-Sic/FAK kinase complexes, noted for involvement in pro-invasive
signaling in cancer. While inhibiting the interaction between
MDA-9/Syntenin and its targets could be fruitful, to date, no
molecular inhibitor of the PDZ domain of MDA-9/Syntenin has been
developed. Fragment-based lead design or fragment-based drug design
(FBDD) is an emerging and useful strategy for the development of
biologically active compounds. Using an NMR guided FBDD approach,
combined with in silica modeling and synthetic medicinal chemistry,
we have now identified first generation PDZ1 MDA-9/Syntenin binding
small molecules, represented by compound 113B7 (herein referred to
as PDZ1i) that binds with micromolar affinity to the PDZ1 domain of
MDA-9/Syntenin (FIG. 4A-4E, FIG. 5, FIG. 6, and Table 1). PDZ1i is
long lived in vivo after both IV and IP administration in mice.
These data indicated that PDZ1i was a suitable pharmacological tool
to investigate the role of the PDZ1 domain of MDA-9 in cellular
mechanistic and in vivo efficacy studies.
TABLE-US-00001 TABLE 1 Binding affinity ranking * ID Compound
Structure (K.sub.d values) 30A9 ##STR00033## 4 (197.2 .mu.M) cmpd1
##STR00034## 2 cmpd2 ##STR00035## 3 cmpd3 ##STR00036## 1 cmpd4
##STR00037## 4 cmpd5 ##STR00038## 2 cmpd6 ##STR00039## 1 cmpd7
##STR00040## 2 cmpd8 ##STR00041## 3 cmpd9 ##STR00042## 3 cmpd10
##STR00043## 4 (151.4 .mu.M) cmpd11 ##STR00044## 5 (170.6 .mu.M)
cmpd12 ##STR00045## 3 cmpd13 ##STR00046## 3 cmpd14 ##STR00047## 1
112D11 ##STR00048## 2 (244.7 .mu.M) 112E7 ##STR00049## 1 (944.2
.mu.M) 112E12 ##STR00050## 1 (1610 .mu.M) 112F1 ##STR00051## 1
112F10 ##STR00052## 4 112F11 ##STR00053## 5 (71.76 .mu.M) 112F12
##STR00054## 2 (165.9 .mu.M) 112G1 ##STR00055## 6 (57.3 .mu.M)
112G2 ##STR00056## 4 (146.8 .mu.M) 112G3 ##STR00057## 4 (119.3
.mu.M) 112G4 ##STR00058## 6 (34.3 .mu.M) 112G11 ##STR00059## 2
(263.1 .mu.M) 113B8 ##STR00060## 5 (151.1 .mu.M) 112H9 ##STR00061##
2 (38.33 .mu.M) 113B7 (PDZ1i) ##STR00062## 4 (21.37 .mu.M) 113B9
##STR00063## N.B. 113B11 ##STR00064## 3 113B12 ##STR00065## 1 *
Rank ordering of compound affinity for PDZ1 were estimated based on
chemical shift changes (DPPM) of the backbone amide of residue 147
on the [.sup.15N, .sup.1H] HSQC spectrum of 50 .mu.M PDZ12 caused
by compound at 1:1 molar ratio: 1 for DPPM 0.00-0.02; 2 for DPPM
0.02-0.04; 3 for DPPM 0.04-0.06; 4 for DPPM 0.06-0.08; 5 for DPPM
0.08-0.1; 6 for DPPM above 0.1; N.B. no appreciable binding
detected. Kd values reported in parentheses were obtained by NMR
titration experiments following the chemical shifts of the backbone
amide of residue 147 at different ligand concentrations.
[0587] Here, the efficacy of supplementing radiotherapy by
targeting, both genetically (shmda-9) and pharmacologically
(PDZ1i), MDA-9/Syntenin in GBM is demonstrated. By counteracting
gains in Src, FAK, and EphA2 signaling, MDA-9/Syntenin inhibition
can reduce radiation-induced invasion, as well as radiosensitize
GBM cells, ideal properties to complement radiation treatment.
[0588] MDA-9/Syntenin inhibition leads to radiosensitization and
inhibition of radiation-induced invasion. Prior studies indicate
that MDA-9/Syntenin is an important mediator of glioma progression.
Specifically, MDA-9/Syntenin was shown to be a valuable target in
addressing one of the deadliest aspects of GBM, its propensity to
invade. Since radiation therapy has been demonstrated to induce
invasion in GBM cells, targeting MDA-9/Syntenin could be a useful
approach to complement conventional treatment. Gliomas with higher
expression of MDA-9/Syntenin are more likely to be high-grade, and
patients whose tumors had high levels of MDA-9/Syntenin had a worse
prognosis. With this in mind, possible connections between
MDA-9/Syntenin expression and radiotherapy in glioma were explored,
using the REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT)
database, a publicly available dataset with information on tumor
gene expression, treatment history, and survival (21). In glioma
patients with no history of radiation who then underwent subsequent
radiotherapy, we probed if survival time correlated with
MDA-9/Syntenin expression. Patients whose tumors had higher
expression of MDA-9/Syntenin had significantly shorter survival
(FIG. 1A, p<0.01). Median survival was reduced nearly threefold
in patients with gliomas expressing high levels of MDA-9/Syntenin
(15.2 months) compared to others (43.6 months).
[0589] The impact of inhibiting the expression of MDA-9/Syntenin on
changes in radiosensitivity was explored in vitro. Using GBM cells
expressing a control shRNA, U1242-shcon, or shRNA targeting
mda-9/syntenin, U1242-shmda-9/syntenin, it was found that low
levels of MDA-9/Syntenin radiosensitized GBM in a colony formation
assay (FIG. 1B). Furthermore, proliferation was inhibited in cells
transiently reduced in MDA-9/Syntenin expression via adenoviral
vector Ad.5/3-shmda-9 (FIG. 1C). This indicates that MDA-9/Syntenin
may have a central role in cell survival following radiation
exposure.
[0590] Radiation consistently increases motility and invasion in
GBM cells. Four cell lines were exposed to radiation after
treatment with control adenovirus, Ad.5/3-shcon, or Ad.5/3-shmda-9.
Each of the cell lines received a range of radiation doses, and the
dose that demonstrated maximal radiation gains was used for
MDA-9/Syntenin knockdown studies. As expected, knockdown of
MDA-9/Syntenin levels reduced invasion with no radiation exposure.
After irradiation, each GBM cell line exhibited at least a twofold
invasion gain, while MDA-9/Syntenin knockdown significantly
curtailed these gains (FIG. 2). GBM invasion following radiation
was reduced with MDA-9/Syntenin knockdown to about 33% of levels
observed in controls. This represents a substantial reduction to an
undesirable side effect displayed upon exposure to conventional
radiotherapy.
[0591] Knockdown of mda-9/syntenin inhibits Src and EphA2
activation post-radiation. MDA-9/Syntenin amplifies Src and
NF-.kappa.B signaling in melanoma and glioma. Src is a recognized
enhancer of invasive signals in cancer settings and has
demonstrated involvement in post-radiation signaling. While a
modest increase in MDA-9/Syntenin levels 24 h post-radiation was
observed, a significant increase in activated levels of Src (FIG.
3) was detected. MDA-9/Syntenin knockdown reduced those activation
levels in each of the GBM cell lines tested. EphA2 is a member of
the Eph-receptor family, the largest subfamily of receptor tyrosine
kinases (RTKs), and are involved in numerous cellular processes
including angiogenesis and motility. EphA2 overexpression
correlates with poor prognosis and increased metastasis, is
implicated in the pathogenesis of numerous tumors, including those
of the brain. Importantly, EphA2 has been shown to interact with
Src in invasion signaling, is upregulated following radiation, and
enhances the malignant phenotype in melanoma. Both total and
activated EphA2 levels increased after radiation exposure, yet
MDA-9/Syntenin inhibition reduced these gains effectively in each
case (FIG. 3). Since MDA-9/Syntenin inhibition negated the gains in
Src and EphA2 signaling post-radiation, it can be considered a
valuable target in controlling radiation-induced pathogenesis.
[0592] A small molecule targeting MDA-9/Syntenin inhibits invasion
and radiosensitizes GBM. To develop a deliverable agent that could
effectively inhibit the action of MDA-9/Syntenin in cellular and in
subsequent in vivo studies, a Fragment-based drug discovery (FBDD)
approach was enlisted. FBDD is an important technique that has been
used with success in efforts to develop small molecule inhibitors
of challenging drug targets including those involved in
protein-protein interactions. NMR based screening of an house
assembled fragment library of about 5,000 compounds using
[.sup.15N, .sup.1H] HSQC correlation spectra with .sup.15N-labeled
PDZ1/2 tandem domain from MDA9 resulted in the identification of
two hit compounds (FIG. 4). Chemical shift mapping studies with
these two classes of hits revealed that these molecules interacted
mainly with the PDZ1 domain and in a region at the interface
between the domains, while no viable fragment hits were found
binding to the PDZ2 domain (FIG. 4). A combination of molecular
docking studies and structure-activity relationships (SAR) studies
led to the synthesis of the molecule 113B7 (PDZ1i; FIGS. 4A-4E).
The proposed docked structure of PDZ1i in the PDZ1 domain and
interdomain of MDA-9/Syntenin is shown in FIGS. 4A-4E and FIG. 5,
while the chemical structure of PDZ1i is also shown in FIGS. 4A-4E
and FIG. 5. In agreement with the data with the individual
fragments that compose 113B7, the molecule does not appreciably
bind to the PDZ2 domain of MDA-9, indicating selectivity (FIGS.
4A-4E). The synthetic scheme for the preparation of the PDZ1i is
reported in FIG. 6. Based on this scheme, relatively large scale
amounts of the compound (>500 mg) were synthesized and used in a
variety of cellular and in vivo characterizations, including
pharmacokinetics (PK) and efficacy studies in mice (Table 2). Other
compounds in Table 1 may also be utilized in the practice of
compositions and method described herein and PDZ1i is a preferred
model of this genus of compounds.
TABLE-US-00002 TABLE 2 Pharmacokinetic data for PDZ1i (113B7).
Balb/c mice (n = 3 per group) were injected with 3 mg/Kg and 30
mg/Kg PDZ1i intravenously (IV) or intraperitoneally (IP),
respectively, and the average concentration of intact compound in
plasma was determined via HPLC at the indicated time points. Plasma
Concentration (ng/mL) in mice (n = 3) after IV (3 mg/kg) and IP (30
mg/kg) administration IV Time (h) Mean IV SD CV (%) IP Time (h)
Mean IP SD CV (%) Body 25.1 .+-. 0.173 0.690 Body 24.5 .+-. 0.252
1.03 Weight (g) Weight (g) 0 ND .+-. ND ND 0 ND .+-. ND ND 0.250
29533 .+-. 7387 25.0 0.250 98233 .+-. 10289 10.5 1.00 17733 .+-.
3630 20.5 1.00 69233 .+-. 9757 14.1 2.00 11600 .+-. 985 8.40 2.00
58967 .+-. 7257 12.3 6.00 6113 .+-. 531 8.68 6.00 54600 .+-. 6351
11.6 12.0 3360 .+-. 927 27.6 12.0 42833 .+-. 7129 16.6 24.0 541
.+-. 79.6 14.7 24.0 15133 .+-. 2754 18.2 No. points 3 No. points 3
used for t.sub.1/2 used for T.sub.1/2 C.sub.0 35033 .+-. 9424 26.9
C.sub.max 9 233 .+-. 10289 10.5 (ng/mL) (ng/mL) T (h) 5.06 .+-.
0.285 5.64 T.sub.max (h) 0.250 .+-. 0.000 0.00 AUC 120000 .+-.
17692 14.7 T (h) .42 .+-. 0.585 6.22 (ng h/mL) AUC 124000 .+-.
17349 14.0 AUC 974667 .+-. 127602 13.1 (ng h/mL) (ng h/mL) AUC
1183333 .+-. 180093 15.2 (ng h/mL) Bioavailability (IP/IV) 81.2%
.+-. 10.6% indicates data missing or illegible when filed
[0593] Initial studies with PDZ1i revealed that it effectively
inhibited invasion in T98G and U87 cells (FIG. 7). Moreover, it was
effective in inhibiting MDA-9/Syntenin--induced invasion following
MDA-9/Syntenin overexpression (FIG. 7). The ability of PDZ1i to
radiosensitize glioma cells was tested. Immortalized astrocytes,
ImPHFA, and the GBM cell line U87 to 0, 2, were exposed to 5 Gy
radiation after a two hour pretreatment with PDZ1i. As expected,
normal astrocytes showed significant radiosensitivity following
radiation exposure, yet PDZ1i treatment did not further
radiosensitize these cells (FIG. 8A). However, U87 cells showed
markedly more radiosensitivity when combined with PDZ1i treatment
Proliferation following radiation was also significantly decreased
in U87 cells that combined radiation and PDZ1i compared to
radiation with control DMSO treatment (FIG. 8B). Finally, U87 cells
were exposed to 2 and 5 Gy of radiation, with or without PDZ1i
pretreatment, which increased the ability of these cells to invade,
while PDZ1i pretreatment abolished these invasion gains (FIG. 8C
and FIG. 8D).
[0594] PDZ1i inhibits EGFRvIII-driven signaling in GBM and reduces
MMP secretion. EGFR amplification and mutation is both a common and
important alteration in GBM. A particular mutation of this
receptor, EGFRvIII, is often co-expressed along with EGFR
amplification in GBM. Recent data has demonstrated that EGFRvIII
and FAK interact as part of a complex to mediate EGFRvIII-mediated
MAPK activation. Given the demonstrated role of MDA-9/Syntenin in
enhancing downstream signaling of the FAK/Src complex in melanoma,
we determined if PDZ1i could inhibit EGFRvIII signaling in GBM. U87
cells stably expressing EGFRvIII, U87-EGFRvIII (FIG. 9), were
treated with PDZ1i prior to radiation. In both radiated and
non-radiated cells, phospho-EGFR was significantly reduced in the
presence of PDZ1i. FAK activation was enhanced after radiation, and
this activation was nullified by PDZ1i treatment (FIG. 9).
Downstream, it was observed that NF-kB activation, enhanced
following radiation, was reduced in the presence of PDZ1i. These
results show that PDZ1i disrupts EGFRvIII-FAK signaling, which
ultimately reduces the observed post-radiation gains in NF-kB
activation.
[0595] Secreted factors can significantly impact cancer cell
invasion, and MDA-9/Syntenin can affect the secretion of important
factors such as MMP2 and VEGF in glioma. Radiation can enhance the
release of important enzymes, including those involved in invasion
such as the matrix metalloproteinase family (MMP). We therefore
determined if PDZ1i could affect the secretion of invasion-related
proteins following radiation therapy. U1242 cells were treated for
2 hours prior to radiation therapy, and their media collected after
48 hours. Several MMP family members showed a significant increase
in expression following radiation therapy, including MMP2 and MMP9.
PDZ1i treatment reduced the levels of these enzymes following
radiation therapy (FIG. 10), an important aspect of reducing
radiation-induced invasion gains. Additionally, radiation therapy
increased the expression of several Cathepsin family members as
well as ADAM9, while PDZ1i eliminated these gains (FIG. 10).
Cathepsin family proteases modulate tumor invasion and metastasis
in a variety of malignancies including melanoma and breast cancer
metastatic to the brain. Notably, the expression of this family is
important in microenvironment-mediated chemo- and radioresistance
and in facilitating supportive tumor stroma.
[0596] A critical property of a potential therapeutic agent is its
stability in the circulation and bioavailability in vivo. Hence,
pharmacokinetics (PK) studies were performed administering 3.0
mg/Kg (IV) and 30.0 mg/Kg (IP) in mice (n=3) and compound
concentration in serum were measured at various times (15 min to 24
hrs after administration of the drug). Noteworthy were the lack of
adverse signs of toxicity during the experiment, the very slow
clearance with T.sub.1/2>9 hr in each route and the 80%
bioavailability for the compound administered IP versus IV (FIG.
11). Based on these data, 1-3 weekly doses of the drug I.P at 30
mg/Kg would result in an effective dose for achieving a constant
inhibition of the target. Additionally, when treating glioma
pharmaceutically, the ability for a compound to cross the blood
brain barrier (BBB) is paramount. We did not anticipate issues
inhibiting brain exposure as it is known that mice with GBM have a
leaky blood brain barrier. Prior to in vivo efficacy studies, we
evaluated the ability of the compound to cross the blood brain
barrier using a target-based surrogate cellular assay. This method
is preferred to direct in vivo measurements of compound
concentration in the brain because of low sensitivity of the
detection methods, and most importantly because of the presence of
drug in various vascular spaces, i.e., the residual blood of the
brain. Accordingly, we used human brain micro vascular endothelial
cells (HBMEC) seeded with PDZ1i in the upper chamber of transwell
inserts placed on top of wells containing GBM6 cells. After 24 h,
we assessed the invasive ability of the treated GBM6 cells compared
to pretreated controls. PDZ1i effectively crossed the HBMEC barrier
to inhibit invasion in GBM6 cells comparably to the preheated
control with no barrier (FIG. 12). Taken together, these results
suggest that PDZ1i is a potent inhibitor of invasion with
radiosensitizing effects and favorable properties for CNS
treatment. Thus, we moved our assessment of PDZ1i as a therapeutic
agent into in vivo models of GBM.
[0597] PDZ1i is effective in reducing tumor invasion in an animal
model of GBM. To test the efficacy of PDZ1i, we used an orthotopic
xenograft model with GBM6 cells that were preheated with either
DMSO or PDZ1i and injected intracranially to form tumors. Seven d
post injection tumor and brain tissue were isolated for sectioning.
Tumor cells treated with PDZ1i developed noticeably smaller and
more demarcated neoplasms at this time point compared to controls
(FIG. 13). Separately, untreated cells were injected and tumors
established, then mice were treated IP with either DMSO or PDZ1i
(30 mg/kg) three times per wk for two wk. Survival was
significantly increased in treated mice compared to controls (FIG.
13), and tumors were well circumscribed and less infiltrative on
analysis than those of control treated mice (FIG. 13). Similar to
previous studies, knocking down MDA-9/Syntenin expression in an in
vivo model, PDZ1i was effective in reducing tumor invasion and
extending survival in an animal model of GBM.
[0598] PDZ1i combined with radiation extends survival and reduces
GBM invasion in vivo. We next investigated the combination of PDZ1i
with radiotherapy in vivo. U1242-Luc cells were injected
intracranially under anesthesia and formed tumors, confirmed by
imaging at 7 d post-injection. At this point, mice were randomized
to 4 groups: DMSO treatment, PDZ1i treatment (30 mg/kg), DMSO+IR,
and PDZ1i+IR. Treatment began on 11 d post-injection and mice
receiving radiation were treated with 2.5 Gy for 4 consecutive d.
PDZ1i was administered 2 h prior to radiation treatment on each of
the 4 treatment d (FIG. 14A). Control treated mice had an average
survival of 41.3 d, while PDZ1i treatment extended that to 54.7 d.
Radiation alone extended survival to 62.8 d, while PDZ1i
treatment+IR therapy led to survival of 78.8 d (FIG. 14B). Finally,
we asked if there were changes in tumor morphology and invasion
pattern between treatment groups. To investigate this, brain tissue
sections were collected and H & E stains were analyzed. Indeed,
U1242-Luc cells developed diffusely infiltrating tumors in control
groups. Tumor margins were slightly more demarcated in groups
treated with PDZ1i (FIG. 14C). Radiation treatment led to generally
smaller, yet still diffuse tumor patterns, which frequently crossed
the midline of the brain with invasive outgrowths. The combination
of PDZ1i with radiation led to tumors with markedly more
circumscribed margins, less invasive outgrowths, and less spread to
the leptomeninges (FIG. 14). Analysis of tumor sections from these
treatment groups indicated that activated forms of EGFR, FAK, and
Sic correlated with PDZ1i treatment status. Those mice treated with
radiation only showed marked increases in activation of these
proteins, while groups that underwent treatment with PDZ1i showed
decreased signal intensity compared both to control and
radiation-treated groups. Collectively, this data demonstrates that
targeting MDA-9/Syntenin is a viable approach to enhancing
conventional radiation treatment
[0599] MDA-9/Syntenin is an important mediator of the
post-radiation signaling process, eliminating the invasion
enhancement induced by radiation. The combination of PDZ1i and
radiotherapy in a model of GBM showed evidence of improvement over
each method alone. In vivo, important interactions occur within the
tumor microenvironment, including extracellular communication
between tumor cells, but also between tumor cells and normal cells,
such as endothelial cells. Therefore, PDZ1i has positive
therapeutic effects on both tumor and normal cells within the tumor
microenvironment
[0600] MDA-9/Syntenin targeting is a viable approach for combating
radiation-induced invasion by radiosensitizing GBM. Development of
a targeted inhibitor leads to a reduction in phenotypes enhanced by
MDA-9/Syntenin, and is effective when combined with conventional
radiotherapy in vivo. The present study highlights a first in class
MDA-9/Syntenin PDZ1 inhibitor with profound anti-invasive effects,
good stability without overt toxicity in vivo, an ability to pass
the blood brain barrier and the capacity to both radiosensitize and
block invasion gains of radiation in aggressive GBM cells in
vivo.
Materials and Methods
[0601] Reagents, plasmids, adenoviruses and stable cell lines.
PDZ1i (113B7)
(N-(2,5-dimethyl-4-(3-(5-phenyl-1,3,4-oxadiazol-2-yl)propanamido)-
phenyl)-8-oxo-5,6,7,8-tetrahydro-4H-cyclopenta[d][1,2,4]triazolo[1,5-a]pyr-
imidine-2-carboxamide) was synthesized from
3-(5-phenyl-1,3,4-oxadiazol-2-yl)propanoic acid (418 mg, 1.92
mmol), HATH (875 mg, 2.30 mmol), and DIEA (0.83 mL, 4.8 mmol) in
DMF (10 mL) (room temperature for 24 h). PDZ1i was purified via a
C18 column (Combi-Flash R.sub.f, Teledyne ISCO), using
acetonitrile-water as an eluent. This compound was used for all
subsequent cellular and in vivo studies, including PK and efficacy
studies (Table 2).
[0602] Small hairpin RNA for mda-9/syntenin (shmda-9), plasmids
pAd-5/3-shmda-9 or pAd.5/3-shcon were constructed with
pSilencer.TM. hygro expression vectors according to the
manufacturer's protocol (Ambion Inc. TX) as previously described
(1), and the resultant plasmids were cleaved with PacI to release
the recombinant adenovirus genomes and then transfected to HEK-293
cells to rescue the corresponding Ad.5/3-based vectors. The rescued
viruses were upscaled using HEK-293 cells and purified by cesium
chloride double ultracentrifugation using standard protocol and the
titers of infectious viral particles were determined by plaque
assay using HEK-293 cells as described (2).
[0603] Cell Lines, Cell Culture, and Treatments.
[0604] Human malignant glioma cells U87, U251, and T98G as well as
grade III astrocytoma lines Sw1088 and Sw1783 were purchased from
the American Type Culture Collection (Manassas, Va.). Human primary
GBM6 cells were described previously and were provided (3). These
and U1242 cells (4) were cultured in DMEM+F12 supplemented with 10%
FCS in a 37.degree. C. incubator supplemented with 5% CO.sub.2.
Primary human fetal astrocytes (PHFA) were obtained from pre-term
abortions as previously described with IRB approval, and
h-TERT-immortalized primary human fetal astrocytes (IM-PHFA) were
produced and cultured as described (1). All cells were routinely
screened for mycoplasma contamination. Cells were treated with
PDZ1i at the indicated concentrations 2 h prior to irradiation.
Irradiations were done using an MDS Nordion Gammacell 40 research
irradiator with a 137-Cs source delivering a dose rate of 0.896
Gy/min.
[0605] Antibodies and Reagents.
[0606] Antibodies against FAK, phospho-FAK (Tyr 576/577), IGF-1R,
phospho-IGF-1R (1135/36), p38MAPK, phospho-p38MAPK (Thr180/Tyr182),
p65, and phospho-p65(S536), Sic, and phospho-Src (Tyr 416), were
obtained from Cell Signaling (Beverly, Mass.). .beta.-actin and
.alpha.-Tubulin were obtained from Abeam (Cambridge, Mass.), and
MDA-9/Syntenin from Abnova (Taipei, Taiwan). Fibronectin was
purchased from Sigma-Aldrich (St. Louis, Mo.).
[0607] Preparation of Whole-Cell Lysates and Western Blotting
Analysis.
[0608] Preparation of whole-cell lysates and Western blotting
analysis was performed as described (5). For densitometry
evaluation, X-ray films were scanned and analyzed with ImageJ
software (NIH).
[0609] Extraction of Total RNA and Real-Time PCR.
[0610] Total RNA was extracted from cells using the QIAGEN
miRNAeasy Mini Kit (QLAGEN, Hilden, Germany). cDNA preparation was
done using ABI cDNA synthesis kit (Applied Biosystems, Foster City,
Calif.). Real-time polymerase chain reaction (RT-PCR) was performed
using an ABI ViiA7 fast real-time PCR system and Taqman gene
expression assays according to the manufacturer's protocol (Applied
Biosystems, Foster City, Calif.).
[0611] Immunohistochemistry.
[0612] A portion of the frozen tumor specimen was fixed in
phosphate-buffered formalin and preserved as paraffin sections
following standard procedure for the maintenance of histological
structure. Paraffin-embedded sections were dewaxed and rehydrated
through incubations in xylene and a gradient series of alcohol.
Antigen retrieval was processed in 10 mM citric acid (pH 6.0) with
microwave treatment for 20 min. Endogenous hydrogen peroxidase was
quenched with 3% H.sub.2O.sub.2 for 20 min. After blocking
non-specific binding sites with 5% normal sera, the sections were
incubated overnight with antibody. The sections were incubated with
appropriate biotinylated secondary antibody and subsequently with
ABC-peroxidase (Vector Elite, Vector laboratories, Burlingame
Calif.). Colorimetric reactions were developed by incubation in DAB
substrate (0.02% DAB, 0.005% hydrogen peroxide), and counterstained
with 10% Harris' hematoxylin. Hematoxylin & eosin staining was
conducted following a standard protocol (6). The images were
analyzed under the Olympus BX41 microscope system equipped with
DP25 digital camera and software. Tissue microarray CNS2081 was
purchased from US Biomax (Rockville, Md.).
[0613] Invasion and Migration Assays.
[0614] Invasion was measured using 24-well BioCoat cell culture
inserts (BD Biosciences, Bedford, Mass.) with an 8-.mu.m-porosity
polyethylene terephthalate membrane coated with Matrigel Basement
Membrane Matrix (100 .mu.g/cm.sup.2). Briefly, the Matrigel was
allowed to rehydrate for 2 h at room temperature by adding warm,
serum-free DMEM. The wells of the lower chamber were filled with
medium containing 10% fetal bovine serum. Cells (5.times.10.sup.4)
were seeded in the upper compartment (6.5-mm membrane size) in
serum-free medium. The invasion assay was done at 37.degree. C. in
a 5% CO.sub.2 humidified incubator for 18 h. At the end of the
invasion assay, filters were removed, fixed, and stained with the
Diff-Quick Staining kit (IMEB, San Marcos, Calif.). Cells remaining
on the upper surface of the filters were removed by wiping with a
cotton swab, and invasion was determined by counting the cells that
migrated to the lower side of the filter using at least 5 fields
per insert at 100.times. magnification. Wound healing scratch
motility assays were done as described previously (6). Data from
triplicate experiments were expressed as mean.+-.95% confidence
interval (CI).
[0615] Survival and Viability Analysis.
[0616] Clonogenic radiosurvival experiments were carried out as
described previously (4, 7). Briefly, cells were diluted, seeded on
6-cm dishes, and after the cells attached, treated with DMSO or
PDZ1i (12.5, 25, or 50 .mu.M) in the medium for 2 h before
irradiation. Cells were incubated overnight, and the medium changed
to nondrug containing medium 16 h postirradiation. Cells were
incubated further for 14 days, stained with 2% Giemsa solution, and
colonies consisting of 50 or more were counted. Cell viability was
assessed through MTT analysis as described previously (8).
[0617] Conditioned Media (CM) Isolation and Analysis.
[0618] CM was harvested from an overnight culture of plated cells
in serum-free DMEM+F12 media, filtered with 0.2 .mu.M filters and
further concentrated 10-fold on an Amicon Ultra centrifugal
filter--3K (Millipore, Billerica, Mass.). Protein levels were
analyzed via the Proteome Profiler Human Protease Array Kit
(R&D Systems, Minneapolis, Minn.). Relative quantification was
performed by densitometry evaluation. X-ray films were scanned and
analyzed with ImageJ software (NIH).
[0619] Virus Infections and Reporter Assays.
[0620] Viral infection conditions and protocols were performed as
delineated previously (6). Luciferase reporter assays were
performed using 2.times.10.sup.5 cells infected with either
Ad.5/3-vec or Ad.5/3-mda-9. Twenty-four h post-infection, cells
were transfected with a NF-.kappa.B-responsive luciferase reporter
construct with LipofectAMINE 2000 as described (6, 9). Forty-eight
h after transfection, cells were trypsinized and plated on
fibronectin-coated surfaces (10 .mu.g/mL) in serum-free medium for
1 h. Cell lysates were harvested and luciferase activity was
measured using a Dual-Luciferase Reporter Assay system (Promega,
Madison, Wis.) according to the manufacturer's instructions.
Luciferase activity was normalized by Renilla activity and data
represent the average of triplicates+S.D.
[0621] Intracranial Implant of Cells in Nude Mice.
[0622] Athymic female NCr-nu/nu mice (NCI-Fredrick) weighing
.about.25 gm were used for this study. Mice were maintained under
pathogen-free conditions in facilities approved by the American
Association for Accreditation of Laboratory Animal Care and in
accordance with current regulations and standards of the U.S.
Department of Agriculture, Washington, D.C., the U.S. Department of
Health and Human Services, Washington, D.C., and the NIH, Bethesda,
Md. At least S mice per group were utilized with duplicate
replications for minimum total of 10 mice per group. Mice were
anesthetized through i.p. administration of ketamine (40 mg/kg) and
xylazine (3 mg/kg), and immobilized in a stereotactic frame
(Stoelting, Wood Dale, Ill.). A 24-gauge needle attached to a
Hamilton syringe was inserted into the right basal ganglia to a
depth of 3.5-mm and then withdrawn 0.5-mm to make space for tumor
cell accumulation. The entry point at the skull was 2-mm lateral
and 1-mm dorsal to the bregma. Intracerebral injections of
1.5.times.10.sup.4 cells in 2 L per mouse were completed over 10
min using an automated injector (Stoelting, Wood Dale, Ill.). The
skull opening was enclosed with sterile bone wax and the skin
incision was closed using sterile surgical staples. Post-sacrifice,
the tumors were resected, weighed, and preserved for IHC
staining.
[0623] Database Mining.
[0624] The REpository for Molecular BRAin Neoplasia DaTa
(REMBRANDT) (rembrandt.nci.nih.gov) database was accessed and mined
for all Astrocytoma and GBM patients (10). These were filtered for
those with no previous history of radiation therapy, but had record
of undergoing subsequent radiation. This population was stratified
for SDCBP (mda-9/syntenin) expression. The survival data for the
resulting groups was downloaded and analyzed via GraphPad
Prism.
Example 2. Suppression of Prostate Cancer Pathogenesis Using an
MDA-9/Syntenin (SDCBP) PDZ1 Small Molecule Inhibitor
[0625] Metastasis is the primary determinant of death in patients
with diverse solid tumors and MDA-9/Syntenin (SDCBP), a
pro-metastatic and pro-angiogenic gene, is a primary contributor to
this process. Here we show that through physical association with
IGF-1R, MDA-9 activates STAT3 regulating prostate cancer
pathogenesis. MDA-9 contains two highly homologous PDZ domains,
which have been difficult to drug, but are predicted to interact
with a plethora of proteins, many of which are central to the
cancerous process. Using fragment-based drug discovery (FBDD)
guided by NMR spectroscopy, an MDA-9 PDZ1 domain-targeted small
molecule antagonist (PDZ1i) has been identified which is
well-tolerated in vivo, has significant half-life (t.sub.1/2=9 h)
and displays substantial preclinical in vivo activity. PDZ1i blocks
tumor cell invasion and migration in vitro, and metastasis in vivo.
Hence, MDA-9 PDZ1 target-specific small molecule inhibitors have
been developed for therapy of prostate and other cancers expressing
elevated levels of MDA-9.
[0626] MDA-9 Expression is Elevated in PC and Promotes
Invasion.
[0627] Recent computational analysis identified mda-9 mRNA
overexpression in PC compared to normal prostate.
Immunohistochemistry (IHC) of MDA-9 protein expression in a tissue
microarray (TMA) containing 9 adjacent tumor tissue and 88 human
prostate tumor samples of different stages supported the genomic
profiling data. Compared to adjacent normal tissue, MDA-9
expression was noticeably increased in PC. mda-9 is elevated at
both mRNA and protein levels (E2 FIG. 15A) in different human PC
cell lines in comparison with non-tumorigenic prostate epithelial
cells. Expression is significantly up-regulated in M12, a
metastatic variant of P69 (a normal immortalized prostate
epithelial cell line), suggesting an association of MDA-9 with
metastasis, which was supported further by in vitro invasion assays
using gain and loss of function approaches (FIG. 15B and FIG. 1C).
In addition, higher expression of MDA-9 was evident in mesenchymal
ARCaPM (metastatic variant) cells in comparison with epithelial
variant ARCaPE cells, providing additional confirmation of the
potential relevance of MDA-9 in metastasis. MDA-9 is also
overexpressed in M2182 cells, which are tumorigenic but
non-metastatic P69 variant cells suggesting that in addition to a
role in metastasis, MDA-9 might also contribute to tumorigenic
phenotypes. A direct role of MDA-9 in PC metastasis was evident in
vivo where stable knockdown of mda-9 using shmda-9 in ARCaPM cells
decreased metastatic potential and increased survival time, as
compared with parental ARCaP-M cells (FIG. 15D). MDA-9 expression
was also elevated in prostate adenocarcinomas that developed in
6-month old and older Hi-myc mice, a spontaneous prostate cancer
transgenic mouse model.
[0628] MDA-9 Regulates PC Invasion Through STAT3 Activation.
[0629] A large percentage of tumor-derived cell lines as well as
human tumors, including PC, express a constitutively activated
STAT3 protein. To determine the effect of MDA-9 expression on STAT3
activity, STAT3 reporter constructs from SA Bioscience (Valencia,
Calif.), which encode a firefly luciferase reporter gene under the
control of a minimal CMV promoter and tandem repeats of the SIE
transcriptional response element, were utilized. These constructs
monitor both increases and decreases in the transcriptional
activity of STAT3-containing dimers, and hence the activity of the
STAT3 signaling pathway. STAT3 activity (measured by phosphorylated
(Tyr705) STAT3) was significantly higher in 4 aggressive PC cells,
which correlated with MDA-9 levels (FIG. 16A and FIG. 15A for
MDA-9). Further evidence for mda-9-mediated STAT3 activation was
documented by phosphorylated STAT-3 levels detected by western
blotting analysis (FIG. 16B), and by co-transfection of a STAT3
reporter with mda-9 or shmda-9 in primary immortal normal prostate
epithelial (RWPE-1), PC-3 and DU145 cells (FIGS. 16C-16E).
Moreover, the anti-invasive activity of MDA-9 was rescued by
overexpressing a constitutive active STAT3 in mda-9 knockdown PC
cells (FIG. 16F). Blocking secretion, using Brefeldin A,
significantly reduced STAT3 activity in PC cells indicating the
contribution of a secretory process in activation (FIG. 16G).
MDA-9-mediated STAT3 regulation involved IGFBP-2, a downstream
target of MDA-9 which was expressed at elevated levels in
aggressive PC cells (FIG. 16H) as documented by co-transfection
studies with various combination of plasmids (FIG. 16I and FIG.
16J).
[0630] MDA-9 Physically Interacts with IGF-1R and Activates
STAT3.
[0631] IGF-1R is a tyrosine kinase that can phosphorylate
STAT3.sup.48. IGF-1R, which belongs to the IGF receptor family, is
a transmembrane receptor tyrosine kinase (RTK). In response to
insulin-like growth factor 1 ligand binding IGF-1R is activated via
autophosphorylation (Tyr 980), leading to activation of various
signaling cascades including STAT3. Because IGF-1R can also
potently activate the STAT3 pathway, one could envision a model in
which IGFBP-2 (through binding with IGF-1) might activate STAT3 in
an IGF-1R-dependent manner. To explore this possibility, we treated
RWPE-1 (immortal normal human prostate epithelial cells) with
recombinant IGFBP-2 (hIGFBP-2) in the absence of serum and Western
blotting was performed to determine autophosphorylation,
representing the activation state of IGF-1R. As anticipated, the
presence of IGFBP-2 in the absence of exogenous IGF-1 (not ruling
out the possibility of endogenous- or cells-derived IGF-1 in the
media) activated IGF-1R, which only occurred in the presence of
MDA-9 (FIG. 16K). Enhanced STAT3 activation was also observed in
these samples. These findings support the importance of MDA-9 in
IGF-1R-mediated STAT3 activation. Further experiments revealed that
MDA-9 physically interacts with IGF-1R (FIG. 16L), which might play
an essential role in transmitting the activation signal to STAT3.
To obtain more insight into the potential binding site(s) and the
consequences of this interaction, different deletion mutants of
mda-9 were overexpressed in RWPE-1 cells and IGF-1R activation was
examined (FIG. 16M). Expression of a PDZ1-deleted fragment
(.DELTA.PDZ1) failed to activate IGF-1R (following hIGFBP-2
treatment) indicating that the potential binding site of IGF-1R
resides in the PDZ1 domain of MDA-9. As expected, STAT3 activity
also correlated with IGF-1R activation further confirming that
MDA-9-mediated STAT3 activation is a downstream consequence of
MDA-9/IGF-1R interaction. It is likely that MDA-9/Syntenin and
IGF-1R physically interact and stabilize the functional unit to
activate STAT3 through phosphorylation at tyrosine 705.
Phospho-STAT3 forms a dimer and translocates into the nucleus to
induce various genes that actively participate in PC progression
(FIG. 16N).
[0632] PDZ1i Suppresses Mda-9-Mediated Invasion in PC and Mda-9
Overexpressing Normal Prostate Epithelial Cells.
[0633] Identification and characterization of a novel PDZ1
antagonist, PDZ1i, was described in Example 1. Initial screening
with established cell lines demonstrated that pre-treatment with
PDZ1i (20 .mu.M or 40 .mu.M) significantly inhibited the invasion
of various types of cancer cells (FIG. 17A) including PC (FIG.
17B). It is worth noting that the compound did not show any
toxicity, even when tested at 50 .mu.M. In addition, to assess the
direct effect of PDZ1i on mda-9-mediated invasion, normal immortal
prostate epithelial (RWPE-1) cells were genetically modified to
overexpress mda-9. The parent RWPE-1 cells express minimal levels
of MDA-9 and possess minimal invasive ability. However, the cells
overexpressing mda-9 displayed increased invasion capability which
was significantly attenuated when cells were treated with PDZ1i
(FIG. 17B). The anti-invasive effect of PDZ1i was also tested on
other PC cells including PC-3, DU14S, metastatic variant ARCaPM
(FIG. 17B). These findings establish that PDZ1i can inhibit
invasion by interrupting mda-9-mediated promotion of transformed
(invasive) properties.
[0634] Next, the effect of PDZ1i on MDA-9 and IGF-1R interactions
was determined. DU145 and ARCaPM cells were treated with PDZ1i for
6 h and cell lysates were subjected to co-IP analysis to determine
potential physical interactions between MDA-9 and IGF-1R (FIG.
17C). The same samples were also analyzed for MDA-9 (FIG. 17D) and
Sic interactions. The results confirmed that PDZ1i selectively
inhibits MDA-9/IGF-1R interactions, but not interactions of
MDA-9/Src. These results provide further evidence for the
specificity of this PDZ1i.
[0635] Further studies confirmed that PDZ1i down regulates IGF-1R
and STAT3 activity in both a dose--(FIG. 17E) and time-dependent
(FIG. 17F) manner. Previous studies indicate that IL-6 can activate
STAT3 through IGF-1R in PC.sup.43. PDZ1i's ability to interrupt
IL-6-mediated STAT3 activation was also demonstrated (FIG. 17G).
Although PDZ1i did not effect MDA-9/Src interactions, Sic activity
was reduced (FIG. 17G) suggesting that IGF-1R might play a role in
Src activation. PDZ1i might directly or indirectly down regulate
multiple receptor tyrosine kinases, which is currently being
explored. Finally, consistent with previous observations.sup.16,
51, PDZ1i downregulates both MMP-2 and MMP-9 at both the protein
and activity level, documented by western blotting and zymography,
respectively (FIG. 17H).
[0636] Anti-Angiogenic Role of PDZ1i.
[0637] In this study, the potential impact of PDZ1i on
tumor-derived angiogenesis in a prostate cancer context was
explored. The experimental strategy is outlined in FIG. 18A. Data
analysis from a qPCR-based array highlighted a number gene that
were downregulated as a consequence of PDZ1i-treatment (FIG. 18B).
This observation was also validated in different PC cells by qPCR.
Additionally, since VEGF-A is a potent angiogenic factor and a
downstream target of STAT3, the expression pattern of VEGF-A at
both mRNA and protein levels was also evaluated in different PC
cells, including mda-9 stably expressing RWPE-1 cells (FIG. 18C).
Finally, both in vitro tube formation and in vivo CAM assays
confirmed the anti-angiogenic properties of PDZ1i-treated tumor
cell-derived conditioned media (not shown).
[0638] PDZ1i Inhibits PC Development In Vivo.
[0639] To evaluate potential therapeutic efficacy of PDZ1i four
sets of in vivo experiments were conducted. First, stable
luciferase expressing ARCaPM (ARCaPM-Luc) cells were pre-treated
with PDZ1i and injected intravenously into animals to determine the
effect of the MDA-9 inhibitor in modulating retention and adhesion
of metastatic tumor cells in the lungs. The results showed that
mice inoculated with PDZ1i pre-treated cells had lower cancer cell
concentrations in the lungs than did control mice inoculated with
non-pretreated cells in mice, and the PDZ1i pre-treated cells were
cleared more rapidly from the lungs (within 2 h post-inoculation).
This suggests that pre-treatment with PDZ1i alters the adhesion
ability of potentially metastatic tumor cells, which would
ultimately have a direct effect on development of metastatic
lesions at secondary sites. Moreover, animals injected with
PDZ1i-treated ARCaPM cells survived longer than control animals
treated with vehicle without the small molecule PDZ inhibitor (FIG.
19A). In another set of experiments, an equal number of cells was
implanted into animals using an intravenous (i.v.) tail vein route.
Experimental groups received 30 mg/kg body weight of PDZ1i LP. As
predicted, the treated groups developed significantly fewer lesions
compared to control groups, as detected by bioluminescence imaging
(BLI) ultimately resulting in prolonged survival (FIG. 19B). In
another set of experiments, RM1-Luc cells were injected by the
intracardiac route in syngeneic C57BL/6 mice, which were treated
with PDZ1i LP. (3 injections/first week). The results were similar
to those observed in the nude mice experiments, i.e., PDZ1i
significantly reduced the tumor burden in the lungs and prolonged
survival (FIG. 19C). In another experiment, 8-week old Hi-myc male
mice were injected with tumor cells and then treated LP. with
either PDZ1i (30 mg/kg) or vehicle 9 times for the first three
weeks. Mice were then maintained until 6-months of age, when
adenocarcinomas hilly develop in this transgenic animal model.
Additionally, to understand drug-mediated molecular changes, 48 h
before sacrifice, a single additional treatment with PDZ1i was
given. Both the size and weight of prostates collected from the
PDZ1i-treated groups were significantly less than the control
groups (FIG. 19D). H & E staining of prostate sections
indicated that histologically the prostate from the control
(vehicle-treated) group showed a significant progression to
adenocarcinoma (FIG. 19E), which was not evident in the
PDZ1i-treated groups (FIG. 19F; representative photomicrographs are
presented). Immunostaining for the expression of pIGF-1R and
pSTAT3, two downstream effectors of MDA-9, were also significantly
reduced in PDZ1i-treated animals, validating the in vitro
observations in vivo.
[0640] A major component of cancer progression relates to tumor
heterogeneity, which may be the driving force in defining why only
10% of PC cases progress to lethality. Based on accumulating data
various pathways are being targeted to effect beneficial outcomes
in patients with metastatic PC including tumor vasculature,
androgen receptors, IGF-1 and IL-6 signaling, and cytoprotective
chaperones. However, therapy of advanced PC, particularly when
metastasis to bone has occurred, still remains an unattainable
objective. This example demonstrates that MDA-9/Syntenin (SDCBP)
provides a molecular target for small molecules that intervene in
the invasive and metastatic properties of PC both in vitro and in
vivo, thus providing a novel approach for managing PC.
[0641] The present studies also identify IGF-1R as a new MDA-9
binding partner. Considering this association, experiments focused
on the functional significance of this interaction. Once IGF-1R
binds to MDA-9 it results in the downstream activation of STAT3.
Apart from inflammation, active STAT3 is known to transcriptionally
regulate various genes involved in survival, proliferation,
invasion and angiogenesis. Since, MDA-9 did not show any direct
impact on cell survival or proliferation in PC cells based in vitro
experiment, it is likely that MDA-9-mediated up regulation of
STAT-3 activity is relevant in regulating cell invasion rather than
cell survival, which is experimentally supported in the present
study.
[0642] Considering the key role of MDA-9/IGF-1R interactions in
STAT-3 activation, which has a decisive impact on PC progression,
small molecule inhibitors that bind to MDA-9 were tested for their
ability to specifically interrupt MDA-9/IGF-1R interactions thereby
affecting STAT3 activation and PC invasion. As described in Example
1, a PDZ1-specific binding small molecule, PDZ1i, was identified,
and it was found that, in micromolar doses, PDZ1i modifies the
ability of IGF-R1 to bind to MDA-9. Previous studies demonstrated
that both PDZ domains of MDA-9 are critical for interacting with
c-Src and the present studies show that PDZ1i downregulates Src
activation, thereby negatively influencing further downstream
signaling, including p38 and NF-.kappa.B activation in different PC
cells. c-Src can be activated in multiple ways, either by
autophosphorylation or through various tyrosine kinases including
IGF-1R. In PC cells, PDZ1i-mediated downregulation of c-Src
activation is partially IGF-1R dependent. Another tyrosine kinase,
EGFR, which is also an interacting partner of MDA-9, has been
reported to activate c-Src in PC. However, PDZ1i did not have any
effect on EGFR activation, at least under the conditions used in
this study (data not shown).
[0643] Because of the clinical significance of IGF-1R in PC,
diverse therapeutic approaches have been tested that target this
protein including human monoclonal antibodies and small molecules
to inhibit IGF-1R activity through distinct targeting approaches,
e.g., IGF-1R ligands, blocking ligand binding, inhibiting enzymatic
activity, etc. As documented in the present studies, rather than
targeting IGF-1R directly, PDZ1i advantageously inhibits IGF-1R
activity by perturbing MDA-9/IGF-1R interactions, which may be
restricted to its' role in cancer cells. Additionally, recent
studies demonstrate that MDA-9/Syntenin-1 (SDCBP) knockout mice are
viable.sup.69,70 and in these mice tumor-supporting inflammation is
inhibited and melanoma metastasis is suppressed.sup.70. Thus, the
activity of MDA-9 is dispensible for normal cellular functions and
MDA-9-targeted molecules are more specific to neoplastic cells, an
important prerequisite for drug development.
[0644] In summary, this study demonstrates that the PDZ1 domain of
MDA-9 is "druggable" using small molecule inhibitors that are
unique to this domain of MDA-9 and which affect its interactions
with specific proteins. Such small molecule MDA-9 inhibitors can be
used to treat, prevent or ameliorate cancer development and/or
metastasis.
[0645] Methods
[0646] NMR studies and Synthesis of PDZ1i were performed as
described in Example 1.
[0647] Human Cell Lines.
[0648] M12 and M2182 (progressed prostate cancer cell s obtained
from VCU School of Medicine, Richmond, Va.). Other cells except
ARCaP with its epithelial and metastatic variants, ARCaPE and
ARCaPM, respectively were obtained from ATCC (Manassas, Va.) and
maintained in culture as per ATCC recommendations. ARCaP, ARCaPE
and ARCaPM cells were obtained from Novicure Biotechnology
(Birmingham, Ala.) and maintained in media as recommended by the
provider. Primary immortal prostate epithelial cells, RWPE-1, were
purchased from ATCC. HuVEC (Human Umbilical Vein Endothelial Cells)
were obtained from Lonza (Allendale, N.J.). All cell lines were
routinely checked for mycoplasma contamination by using commercial
kits. Cell lines were purchased from ATCC, an authenticated and
realaible source for human cancer cells. In our study most of our
experiments carried out in RWPE-1, PC-3, DU145 and ARCaPM cells.
All of these cell lines were purchased recently (within the last 3
years) and strictly maintained as per the manufacturer's
recommendation. The other cell lines P69, M12 and M2182 were used
to as panel of prostate cancer cells and described in our previous
publications. Androgen-refractory mouse prostate cancer cell lines
RM1, was provided and was maintained in DMEM as previously
described. The metastatic capability of stable
luciferase-expressing RM1 (RM1-Luc) was previously reported.
[0649] Reagents and Antibodies.
[0650] A list of antibodies used in this study are provided in
supplemental "Methods". All reagents for cell cultures including
media and serum were purchased from Gibco. Recombinant IGFBP-2 was
purchased from R & D Biosystems (Minneapolis, Minn.).
[0651] Gelatin Zymography.
[0652] Gelatin zymography is used to determine the gelatinolydc
activity of MMP-2 and MMP-9 in conditioned media collected from in
vitro cell cultures. Briefly, the cancer cells were treated with
either DMSO or 113B7 (PDZ1i), cultured for 48 h, and then the media
was replaced with fresh serum-free media and cultured for an
additional 24 h. The conditioned media was then collected. Equal
amounts of protein containing conditioned media was electrophoresed
in 8% SDS-polyacrylamide gels containing 1.5 mg/mL gelatin. The
gels were washed three times for 30 min each with 2.3% Triton X-100
solution to remove SDS. The gels were then incubated at 37.degree.
C. overnight in incubation buffer [50 mmol/L Tris-HCl (pH 7.5),
0.05% NaN.sub.3, 5 mmol/L CaCl.sub.2, and 1 .mu.mol/L ZnCl.sub.2].
After incubation the gels were stained with 0.1% Amido black
staining solution and subsequently destained with destaining
solution to visualize the gelatinolydc activities that were
identified as clear zones of lysis against (clear band) a blue
background.
[0653] Tissue Microarray.
[0654] A prostate cancer tissue microarray along with matched
adjacent normal tissues was purchased from Imgenx Corp (Currently
part of Novus Biologicals (Littleton, Colo.)) and stained with
MDA-9 antibody (anti rabbit) from Sigma Aldrich (St. Louis).
[0655] Real Time PCR.
[0656] For qPCR, total RNA were extracted using miRNeasy kits
(Qiagen, Valencia, Calif.) as recommended by the manufacturer and
cDNA was prepared as previously described.sup.27. Quantitative qPCR
was performed using an ABI ViiA7 Cast real-time PCR system and
Taqman gene expression assays according to the manufacturer's
protocol (Applied Biosystems, Foster City, Calif.).
[0657] Constructs and Stable Cell Clones.
[0658] Various vector constructs used in this study were either
cloned by our group (mda-9, shmda-9).sup.27 or purchased from
Adgene (Cambridge, Mass.) (pRc.CMV.Stat3Y703F). For developing
stable luciferase-expressing clones, cells were transfected with
expression vectors and selected with neomycin for approximately 2
weeks. Individual colonies were picked and analyzed for luciferase
expression.
[0659] In Vivo Experiments.
[0660] All in vivo experiments were performed in accordance with
IACUC approved protocols. To determine the effect of PDZ1i on tumor
cell retention in the lungs, we inoculated cohorts of mice (n=S,
each group) via tail vein injection with either vehicle (DMSO) or
test compound (50 .mu.M) pre-treated ARCaPM-Luc (1.times.10.sup.6
cells in 100 .mu.l saline) metastatic PC cells. Luciferase activity
was monitored for differential cellular clearance from the lungs
between 15 min and 5 h by Bio luminescence imaging (Xenogen in vivo
imaging (IVIS) system) (Caliper Life Sciences, Inc., Hopkinton,
Mass.). For the lung experimental metastasis model, a total of
5.times.10.sup.s ARCaPM-Luc cells were injected (in 100 .mu.L PBS)
by intravenous tail vein injection. Treatment began 12 days after
PC cell injection. PDZ1i was given at a dose of 30 mg/kg body
weight in solution containing DMSO, Tween 20 and PBS (10:10:80).
Drug was delivered every alternate day for the first three weeks
(total 9 injections). Mice were periodically observed for any signs
of toxicity. Mice were kept until euthanized as recommended by
IACUC. In other experiments C57BL/6, 1.times.10.sup.5 RM1-Luc cells
(TRAMP-derived prostate cancer cells.sup.76).
[0661] Animals were injected by the intracardiac route to develop
lung metastases. Similar to athymic nude mice studies, experimental
mice received only three doses of PDZ1i within the first week of
treatment. Mice were kept until they required euthanasia. In an
additional experiment, Hi-myc mice (prostate cancer spontaneous
transgenic mouse model).sup.38 were injected with PDZ1i i.p.
starting at 2 months of age and continued for subsequent three
weeks (total 9 injections). Mice were kept until they reached 6
months of age. Prostates were removed, photographed, weighed and
processed for paraffin sectioning. Immunohistochemistry was done as
previously described.sup.27 with the antibodies as indicated.
[0662] Co-Immunoprecipitation.
[0663] Co-Immunoprecipitation was performed as described
previously.sup.27 using kit from Pierce (Pierce Biotechnology,
Rockford, Ill.).
[0664] Invasion Assay.
[0665] Boyden chamber assays were done to investigate the invasive
properties of cancer cells.sup.26, 27. Briefly, cells were
pretreated with PDZ1i or DMSO and plated on the upper chamber.
After 18 h, invasive cells were photographed and analyzed.
[0666] In Vitro Tube Formation and Chorioallantoic Membrane (CAM)
Assays.
[0667] Tube formation and CAM assays were performed as described
previously.sup.21. Briefly, tumor-derived conditioned media were
collected after 24 h of treatment (either DMSO or PDZ1i) and
concentrated. Equal amounts of protein (50 .mu.g) containing
conditioned media were mixed with basal media and incubated with
HuVEC cells on Matrigel layers. Photographs were taken after 6 h.
In CAM assays, DMSO- or PDZ1i-treated tumor cell-derived
conditioned media were implanted on the CAM of 8 day-old fertilized
eggs. Photographs were taken on day 12.4 days after applying
conditioned media.
Example 3: MDA-9/Syntenin (SDCBP) Enhances Epithelial Mesenchymal
Transition in Breast Cancer
[0668] MDA-9/Syntenin (SDCBP) is a scaffold protein that plays a
key role in tumor progression and metastasis in several cancer
indications. We have uncovered a novel mechanism by which MDA-9
enhances metastasis in breast cancer. Epithelial mesenchymal
transition (EMT) is a key step in the process of metastasis and we
have uncovered the mechanism by which MDA-9 mediates EMT in breast
cancer. When the expression of MDA-9 was suppressed in metastatic
mesenchymal breast cancer cell lines, an obvious change in cell
morphology was observed where the cells appeared epithelial like.
Conversely, when MDA-9 was overexpressed in non metastatic
epithelial breast cancer cells, the cell morphology appeared
mesenchymal like. Consistent with these findings, several EMT
markers were altered following modulation of MDA-9 expression.
Additionally, changes in cytoskeletal organization and invasive
abilities were also observed. At the mechanistic level, we found
that MDA-9 upregulated active levels of known EMT modulators--the
small GTPases RhoA and Cdc42 via TGF.beta.1. Further we determined
that MDA-9 interacts with TGF.beta.1 and the PDZ1 domain of MDA-9
is key for this interaction. Finally we verified our observations
using in vivo studies. In an in vivo lung metastasis model,
suppressing the expression of MDA-9 resulted in a decrease in lung
metastasis that could be partially restored by re-expression of
TGF.beta.1. Our findings uncover the importance of MDA-9 in EMT in
breast cancer and identify MDA-9 as a therapeutic target against
metastatic breast cancer.
[0669] Our studies demonstrate for the first time that MDA-9
enhances epithelial mesenchymal transition (EMT) in breast cancer
via interaction with TGF.beta.1. MDA-9 has been reported to
regulate cancer metastasis, however, this is the first time that we
report the role of MDA-9 in EMT in breast cancer. The novel
mechanisms of action that we uncover in our study also serve to
identify therapeutic targets against metastatic breast cancer.
[0670] Considering the pivotal role of MDA-9 in regulating
metastasis, directly targeting MDA-9 expression or its interaction
with TGF.beta.1 using genetic or pharmacological approaches may
provide a unique opportunity to develop targeted therapies against
metastatic disease.
[0671] Most available technology has been unsuccessful in
effectively targeting tumor metastases and hence our findings
provide an important therapeutic intervention option for metastatic
breast cancer.
[0672] Developing a targeted approach or specifically inhibiting
MDA-9 interaction with TGF.beta.1 might overcome this problem.
Since an MDA-9 knockout mouse (lacking mda-9 expression in all
tissues) is viable, targeting MDA-9, which is syntenin-1, would not
be predicted to be toxic. Indeed, we have generated inhibitors of
MDA-9 that target the PDZ1 domain of MDA-9 and alter
protein-protein interactions which results in suppression of tumor
invasion, tumor cell attachment, tumor angiogenesis and
metastasis.
[0673] Metastatic breast cancer therapy remains a challenge in the
clinic. We identified MDA-9/Syntenin (syndecan binding protein,
SDCBP) as a key player in enhancing metastasis and identified its
detailed molecular mechanism of action. This understanding of the
role of MDA-9 as well as its mechanism of action has uncovered
several therapeutic options to specifically target metastatic
breast cancer.
[0674] MDA-9/Syntenin (SDCBP) modulates small GTPases RhoA and
Cdc42 via transforming growth factor .beta.1 to enhance
epithelial-mesenchymal transition in breast cancer.
Epithelial-mesenchymal transition (EMT) is one of the decisive
steps regulating cancer invasion and metastasis. However, the
molecular mechanisms underlying this transition require further
clarification. MDA-9/syntenin (SDCBP) expression is elevated in
breast cancer patient samples as well as cultured breast cancer
cells. Silencing expression of MDA-9 in mesenchymal metastatic
breast cancer cells triggered a change in cell morphology in both
2D- and 3D-cultures to a more epithelial-like phenotype, along with
changes in EMT markers, cytoskeletal rearrangement and decreased
invasion. Conversely, over expressing MDA-9 in epithelial
non-metastatic breast cancer cells instigated a change in
morphology to a more mesenchymal phenotype with corresponding
changes in EMT markers, cytoskeletal rearrangement and an increase
in invasion. We also found that MDA-9 upregulated active levels of
known modulators of EMT, the small GTPases RhoA and Cdc42, via
TGF.beta.1. Reintroducing TGF.beta.1 in MDA-9 silenced cells
restored active RhoA and cdc42 levels, modulated cytoskeletal
rearrangement and increased invasion. We further determined that
MDA-9 interacts with TGF.beta.1 via its PDZ1 domain. Finally, in
vivo studies demonstrated that silencing the expression of MDA-9
resulted in decreased lung metastasis and TGF.beta.1 re-expression
partially restored lung metastases. Our findings provide evidence
for the relevance of MDA-9 in mediating EMT in breast cancer and
support the use of MDA-9 as a therapeutic target against metastatic
disease.
[0675] The American Cancer Society estimates that in 2016, about
246,660 women will be diagnosed with breast cancer and
approximately 40,450 women will die of the disease in the United
States (American Cancer Society, Cancer Facts & Figures, 2016).
Despite enhanced early detection, breast cancer is the second
leading cause of cancer-related death among women in the United
States. One of the reasons for this discrepancy is that treatment
options are limited once primary tumors metastasize to distant
areas in the body. Overall prognosis and patient survival are also
adversely affected in metastatic disease. Consequently, it is
imperative to identify relevant therapeutic targets that can
inhibit metastasis of breast cancer. At the molecular level,
epithelial-mesenchymal transition (EMT) enhances invasion and
metastasis of cancer cells. EMT is a well conserved cellular
process during which polarized, non-motile epithelial cells lose
their polarized organization and cell-cell junctions and transition
into motile mesenchymal cells [1, 2]. EMT is now widely accepted as
a mechanism utilized by cancer cells to gain access to distant
areas in the body [2-5]. Identifying distinctive molecules that
regulate EMT and are "druggable" are thus critical to gain control
of metastatic disease.
[0676] Melanoma differentiation associated gene-9 (MDA-9), also
known as syntenin-1 (SDCBP; syndecan binding protein), is a member
of the PDZ-domain containing family and is located on chromosome
8q12 [6]. Initially identified in our laboratory while screening
for genes that were differentially expressed in human melanoma
cells reprogrammed to terminally differentiate [7], MDA-9 has now
been identified as a multifunctional protein involved in diverse
physiological and pathological processes [8, 9]. MDA-9/syntenin
plays an important role in several cellular functions including
regulating cell-cell and cell-matrix adhesion, signal transduction
from the cell surface to the interior through interaction with a
number of proteins, intracellular and secreted lipid trafficking,
and cell surface targeting [6, 9]. Recent studies also implicate
MDA-9 as a key gene involved in cancer stem cell growth and
survival [6, 9]. MDA-9 was also found to play a causative role in
the progression of several different cancer types including
melanoma [10-12], gastric cancer [13], bladder cancer [14],
glioblastoma [15], small cell lung cancer [16], hepatoma [17] and
head and neck cancers [18]. Recently, a study analyzing clinical
patient samples found that MDA-9 expression was higher in patients
with breast cancer and was associated with poor overall patient
outcome [19]. Another study showed that MDA-9 regulates tumor cell
growth in breast cancer [20]. However, the molecular mechanisms
underlying the functional relevance and consequences of MDA-9 in
breast cancer remains largely unexplored.
[0677] In the present study, we evaluated the role of MDA-9 in the
invasive, metastatic and EMT abilities of breast cancer cells. We
assessed the expression pattern of MDA-9 in breast cancer patient
samples and breast cancer cell lines, and examined the impact of
loss-of-function and gain-of-function of MDA-9 expression on
metastatic and non-metastatic breast cancer cells. We further
elucidated the molecular mechanism by which MDA-9 regulates EMT and
metastasis in breast cancer. This is the first study that
identifies the detailed molecular mechanism by which MDA-9
regulates EMT in breast cancer. Overall, our findings show that
MDA-9 could provide a useful therapeutic and diagnostic target for
breast cancer metastasis.
[0678] MDA-9 expression is elevated in human breast cancer.
Metastatic breast cancer continues to pose a formidable problem for
favorable patient outcome [21]. We recently demonstrated that MDA-9
plays a causative role in tumor progression and metastasis in
melanoma [10, 11], urothelial [14], glioblastoma [IS], and head and
neck cancers [18]. To determine the role of MDA-9 in progression
and metastasis of breast cancer, we assessed the expression of
MDA-9 in patient samples and cell lines. A commercially available
tissue microarray of breast cancer patient samples comprising
adjacent normal breast tissue, breast tumor tissue and metastatic
lesions was probed for MDA-9 expression by immunohistochemistry.
Breast tumor tissue and metastatic lesions showed an increased
expression of MDA-9 as compared to normal breast tissue (FIG. 20A).
This finding is in agreement with previous studies and was
performed to verify previously published research to confirm that
MDA-9/syntenin expression was elevated in breast cancer tissues and
metastatic breast cancer cell lines [13, 19, 20]. A clinical study
also found that elevated expression of MDA-9 correlated with
increased metastasis and tumor recurrence in breast cancer patients
[19]. This study showed that both overall survival and disease-free
survival were reduced when MDA-9 expression was elevated [19]. We
also assessed the DNA copy number of MDA-9 in the TCGA database to
assess MDA-9 in a larger cohort of breast cancer patients (FIG.
27). MDA-9 DNA copy number was elevated in breast cancer patients
as compared to normal controls in this larger cohort as well. Next,
we assessed the expression of MDA-9 in a number of non-metastatic
and metastatic human breast cancer cell lines and found that MDA-9
expression was elevated in metastatic cells at both the protein
(FIG. 20B) and transcript level (FIG. 20C). Having validated that
MDA-9 was indeed upregulated in breast cancer and was associated
with increased metastatic incidence, we performed studies to
understand the role of MDA-9 in the metastatic process in breast
cancer.
[0679] Modulating the Expression of MDA-9 in Breast Cancer Cells
Correlates with Changes in Invasive Abilities and Actin
Cytoskeletal Rearrangement.
[0680] One of the hallmarks of cancer and particularly of
metastasis, is the ability to invade into the surrounding basement
membrane [4]. To investigate the importance of MDA-9 in metastatic
breast cancer cells, we silenced MDA-9 expression in metastatic
breast cancer cells, MDA-MB-231 and SUM159, using shRNA targeted to
MDA-9 and non-targeted control (FIG. 21A). Silencing the expression
of MDA-9 caused a dramatic reduction in invasion in both MDA-MB-231
and SUM159 cells (FIG. 21B). Next, we over expressed MDA-9 in
non-metastatic breast cancer cells T47D using an adenovirus
expressing MDA-9 and vector control. Overexpressing MDA-9 in T47D
cells caused an increase in invasive abilities of these cells (FIG.
21B). Reorganization of the cytoskeleton via polymerization and
depolymerization of filamentous actin (F-actin) leads to changes in
cell shape and assists in cell motility [1, 22]. We determined
whether MDA-9 was able to regulate cytoskeletal rearrangement in
breast cancer cells by staining for F-actin using phalloidin.
Silencing the expression of MDA-9 in MDA-MB-231 and SUM159 cells
caused a decrease in stress fibers, while overexpressing MDA-9 in
T47D cells caused an increase in stress fiber formation (FIG.
21C).
[0681] Modulating the Expression of MDA-9 in Breast Cancer Cells
Correlates with Changes in Cell Shape in 2D- and 3D-Culture.
[0682] Modulating MDA-9 expression caused changes in cell shape in
2-dimensional (2D) culture conditions on plastic plates. Silencing
the expression of MDA-9 in mesenchymal metastatic cells MDA-MB-231
and SUM159 caused the cells to appear epithelial-like (FIG. 28).
Conversely, the epithelial T47D cells transitioned to a more
mesenchymal phenotype following over expression of MDA-9 in
2D-culture. Next, the effects of modulating MDA-9 in cells grown in
3-dimensional (3D) culture conditions were assessed. Growing
mammary epithelial cells in 3D-culture on a reconstituted basement
membrane causes the cells to form spheroids that recapitulate
several aspects of glandular architecture in vivo [23]. MDA-9
silenced cells formed compact spherical structures (spheroids) and
lacked the invasive structures produced by the non-targeted control
cells (FIG. 22A). This indicates that MDA-9 silenced cells lose
their ability to invade into the basement membrane and surrounding
matrix. Conversely, when grown in 3D-culture conditions, unlike the
compact spheroids observed in T47D control cells, T47D cells
overexpressing MDA-9 produced projections, indicative of an
increased ability to invade the basement membrane and surrounding
matrix (FIG. 22A).
[0683] Modulating the Expression of MDA-9 in Breast Cancer Cells
Correlates with Changes in EMT.
[0684] Recent studies have identified EMT as the mechanism by which
non-motile epithelial cancer cells progress towards more aggressive
motile and invasive mesenchymal cells [1, 4]. Since MDA-9 enhanced
invasive abilities and we observed a change in cell morphology both
in 2D- and 3D-culture upon modulating the expression of MDA-9,
which is indicative of EMT, we evaluated the role of MDA-9 in EMT
and assessed the expression of several EMT markers in MDA-9
silenced metastatic breast cancer cells and MDA-9 overexpressing
non-metastatic breast cancer cells (FIG. 22B). Silencing the
expression of MDA-9 caused a reduction in mesenchymal markers Slug,
Snail and Zeb1 in both MDA-MB-231 and SUM159 cells. N-cadherin was
also decreased in SUM159 cells. MDA-MB-231 cells are N-cadherin
negative [24]. There was a slight increase in the epithelial marker
ZO-1. Both SUM159 and MDA-MB-231 cells are E-cadherin negative
[24], Conversely, overexpressing MDA-9 in T47D cells resulted in a
decrease in epithelial markers E-cadherin and ZO-1 and an increase
in mesenchymal markers Slug, Snail and Zeb1. T47D cells are
N-cadherin negative [25].
[0685] MDA-9 Modulates the Small Rho GTPoses RhoA and Cdc42 and
Enhances Invasion and Cytoskeletal Rearrangement Via
TGF.beta.1.
[0686] Since the Rho family GTPases, RhoA and Cdc42, are known
modulators of the actin cytoskeleton and play a vital role in EMT
and metastasis in breast cancer [1, 26-28], we assessed the
activity of these GTPases following MDA-9 modulation. We found that
the active levels of RhoA and Cdc42 were downregulated when MDA-9
expression was silenced, while the active levels of RhoA and cdc42
were upregulated when MDA-9 was over expressed (FIGS.
23A-23C--first two bars on each of the graphs).
[0687] One of the key modulators of RhoA and Cdc42 is TGF.beta.1
[29, 30]. To determine whether MDA-9/syntenin might regulate
TGF.beta.1 to modulate RhoA and Cdc42 expression we assessed the
expression levels of TGF.beta.1. We found that TGF.beta.1 levels
were downregulated when MDA-9 expression was silenced while
TGF.beta.1 levels were upregulated when MDA-9 was overexpressed
(FIG. 23D). To determine whether MDA-9 mediates its effects on RhoA
and Cdc42 via TGF.beta.1, we re-introduced TGF.beta.1 in MDA-9
silenced cells and assessed active RhoA and Cdc42 levels (FIGS.
23A-23B). Similarly, we inhibited TGF.beta.1 in T47D cells
overexpressing MDA-9 and assessed the expression of RhoA and Cdc42
(FIG. 23C). To further validate these findings in relation to the
role of MDA-9 in invasion, we found that re-introducing TGF.beta.1
in MDA-9 silenced cells restored the invasive abilities of
MDA-MB-231 and SUM159 cells and caused cytoskeletal rearrangement
(FIGS. 24A-24B). Conversely, inhibiting TGF.beta.1 expression in
MDA-9 overexpressing cells resulted in a decrease in invasive
abilities and cytoskeletal rearrangement (FIG. 24C).
[0688] PDZ1 domain of MDA-9 interacts with TGF.beta.1.
[0689] Next, we endeavored to determine how MDA-9 regulates
TGF.beta.1. To assess whether MDA-9 regulates the transcription of
TGF.beta.1, we performed luciferase reporter assays using
TGF.beta.1 promoter luciferase constructs, but did not find an
increase in luciferase reporter activity. This indicates that MDA-9
did not regulate TGF.beta.1 at the transcriptional level. We
searched the Oncomine database to determine any association between
MDA-9 and TGF.beta.1 in breast cancer patient populations.
Interestingly, we found a dataset that showed that MDA-9 and
TGF.beta.1 were co-expressed in a set of breast cancer patients
(FIG. 29). We also found that Slug (SNAI2), a well-known
EMT-inducing transcription factor, was also co-expressed with MDA-9
and TGF.beta.1, further supporting our overall hypothesis. Next we
determined the DNA copy number of TGF.beta.1 in the same breast
cancer patient cohort that was assessed for MDA-9 DNA copy number
in FIG. 27 (FIG. 30). We observed an association between the DNA
copy numbers of MDA-9 and TGF.beta.1 in the breast cancer patient
samples.
[0690] To validate these findings, we performed
coimmunoprecipitation assays and determined that MDA-9 physically
interacted with TGF.beta.1. First, we performed immunoprecipitation
in SUM159 cells using the MDA-9 antibody and immunoblotted with the
TGF.beta.1 antibody and found that MDA-9 interacts with TGF.beta.1
(FIG. 25A). Next we performed immunoprecipitation in SUM159 control
cells overexpressing TGF.beta.1 and SUM159 cells silenced for MDA-9
expression and overexpressing TGF.beta.1 in order to easily pull
down TGF.beta.1 using the TGF.beta.1 tag and immunoblotted using
the MDA-9 antibody and found that MDA-9 interacted with TGF.beta.1
(FIG. 25B). The MDA-9-TGF.beta.1 interaction was decreased in MDA-9
silenced cells, further validating our observations. Next, we
determined the region of MDA-9 that was involved in the interaction
with TGF.beta.1. FIG. 25C shows the full length MDA-9 construct and
a PDZ1 deleted version of MDA-9. Deletion of the PDZ1 domain
disrupts the interaction between MDA-9 and TGF.beta.1 (FIG. 25D).
This indicates that the PDZ1 domain is essential for its
interaction with TGF.beta.1.
[0691] Silencing the expression of MDA-9 in metastatic breast
cancer cells causes a decrease in lung metastases in vivo, which
could be partially reversed by TGF.beta.1 restoration. To further
validate our findings that MDA-9 was able to enhance the metastatic
potential of breast cancer, we performed in vivo lung metastasis
studies. We developed MDA-MB-231 control, MDA-MB-231 shMDA-9,
MDA-MB-231 control TGF.beta.1 and MDA-MB-231 shMDA-9 TGF.beta.1
cells stably expressing luciferase (FIG. 26A). By incorporating
luciferase into these MDA-MB-231 cell lines we were able to monitor
development of lung metastasis via bioluminescent imaging (BLI).
MDA-MB-231 control luciferase cells were able to colonize the lungs
and establish metastases following introduction into athymic mice
intravenously via the tail vein (FIG. 26B). MDA-MB-231 shMDA-9
luciferase cells however showed a dramatic reduction in the ability
to colonize and establish metastases in the lungs. As would be
expected, MDA-MB-231 control TGF.beta.1 luciferase cells were also
able to colonize the lungs and establish lung metastases.
Importantly, re-expressing TGF.beta.1 in MDA-MB-231 cells silenced
for the expression of MDA-9 caused a partial restoration of lung
metastases (FIGS. 26C-26D). Further, tumor cells were harvested
from the lung metastases that developed in the athymic mice and
re-grown in culture. These cells were evaluated for expression of
MDA-9 and TGF.beta.1. MDA-MB-231 cells that were silenced for MDA-9
expression continued to show very low MDA-9 expression and
TGF.beta.1 expressing cells showed TGF.beta.1 expression (FIG.
26E). FIG. 26F shows H&E staining of the tumor sections showing
presence of lung metastases. The entire lungs were colonized by
both MDA-MB-231 control luciferase cells and MDA-MB-231 control
TGF.beta.1 luciferase cells. However only a few small lung
metastases were observed in the H&E sections of the lungs
injected with MDA-MB-231 shMDA-9 cells. Re-expressing TGF.beta.1 in
MDA-MB-231 cells allowed these cells to form larger lung metastatic
lesions as observed in the H&E section.
[0692] While widespread awareness regarding breast cancer has
facilitated early detection and improved overall patient outcomes,
patient prognosis is adversely affected when breast cancer
metastasizes to distant areas in the body. Hence, identifying novel
therapeutic targets that inhibit EMT and metastasis are key
elements in effectively targeting metastatic breast cancer. We
report here that MDA-9 is upregulated in breast cancer as well as
metastases and plays a key role in EMT induction in breast cancer
cells. We further provide detailed mechanistic insights into the
signaling mediated by MDA-9 to enhance EMT.
[0693] MDA-9 is a scaffold protein with multiple diverse roles in
tumorigenesis, particularly, in tumor invasion and metastasis.
MDA-9 was found to be over expressed in a number of human cancers
including melanoma [10-12], gastric cancer [13], bladder cancer
[14], glioblastoma [15], small cell lung cancer [16], hepatoma
[17], head and neck cancers [18] and breast cancer [19]. Due to its
seminal role in several cancers, researchers have focused on
dissecting the signaling mechanisms regulated by MDA-9. In the
various cancer types evaluated, MDA-9 orchestrates cancer
attributes via its interaction with key binding partners including
several oncogenic proteins [9, 31]. In melanoma cells, MDA-9
colocalizes with focal adhesion kinase (FAK), a key component of
integrin-mediated signaling pathways, and increased phosphorylation
of FAK, c-Jun-NH2-kinase (JNK) and p38 MAPK [10]. MDA-9 was also
shown to interact with c-Src, which enhanced FAK/c-Src complex
formation and activated c-Src [11]. Studies using glioblastoma
cells also showed that MDA-9 increased the activation of c-Src, p38
MAPK and nuclear factor kappa B (NF-F1B), which enhanced expression
of matrix metalloproteinase 2 (MMP2) and the secretion of
interleukin-8 (IL-8) [IS]. In urothelial cancer cells, MDA-9
interacts with EGFR and enhanced the expression of EGFR, AKT,
phosphoinositide 3-kinase (PI3K) and c-Src [14]. In head and neck
squamous cell carcinoma, MDA-9 colocalized with VEGFR1 and
regulated the expression of SPRR1B and VEGFR1. Growth regulatory
molecules including Cyclin D1, CDK4, STAT3, PI3K and CTNNB1 were
also modulated by MDA-9 [18].
[0694] The detailed mechanism by which MDA-9 regulates invasion and
metastasis of breast cancer remains largely unknown. The findings
from our study provide insights into the signaling mechanisms
regulated by MDA-9 in breast cancer. We show that MDA-9 expression
correlated with invasiveness and metastatic capabilities of breast
cancer cells. Loss-of-function and gain-of-function studies
confirmed the relevance of MDA-9 in EMT, invasion and cytoskeletal
rearrangement and helped elucidate the molecular mechanisms of
action of MDA-9. We demonstrate that MDA-9 regulates the small
GTPases RhoA and Cdc42 via TGF.beta.1. Researchers have shown that
treating prostate cancer cells with TGF.beta.1 induced rapid
formation of lamellipodia and cytoskeletal rearrangements [32].
Importantly, this response to TGF.beta.1 was independent of
canonical Smad signaling and required the activity of small Rho
GTPases Cdc42 and RhoA [32]. This study, albeit in a different
cancer indication, supports our findings and suggest that MDA-9
could act upstream of TGF.beta.1 to mediate cytoskeletal
rearrangements.
[0695] We further show that MDA-9 could interact with TGF.beta.1.
We also observed that MDA-9 and TGF.beta.1 were co-expressed in
breast cancer patient samples in the TCGA database using Oncomine
(FIG. 29). Re-introducing TGF.beta.1 in MDA-9-silenced cells caused
a partial restoration of invasive abilities. Similarly, restoring
TGF.beta.1 expression in MDA-9-silenced cells caused a partial
restoration of lung metastases in vivo. Interestingly, a study in
A549 lung carcinoma cells showed that MDA-9 might also prevent
caveolin-1-mediated internalization of TGF.beta.R1 leading to
enhanced canonical TGF.beta.1 signaling [33]. These findings
provide evidence for another layer of regulation of TGF.beta.1
signaling by MDA-9 supporting our overall hypothesis. MDA-9 is a
known scaffold protein and the MDA-9-TGF.beta.1 interaction might
also serve to stabilize the TGF.beta.1 protein. Further
investigation into the mechanism by which MDA-9 regulates
TGF.beta.1 is ongoing. We also observed that MDA-9 and Slug (SNAI2)
were co-expressed in breast cancer patient samples in the TCGA
database using Oncomine (FIG. 29). A very recent study in lung
adenocarcinoma showed that MDA-9 interacts with Slug and regulates
invasion and metastasis, further validating our findings [34],
Further, studies have identified a link between triple negative
breast tumors and the occurrence of EMT [35, 36]. From the
observations in FIG. 20B and FIG. 20C and the understanding we have
gained regarding the role of MDA-9 in breast cancer in this paper,
one can speculate that there might be an association between the
expression of MDA-9 and triple negative breast cancer.
[0696] Interestingly, integrin .beta.1 has been shown to play a
role in aiding TGF.beta.1-mediated non-canonical signaling
including downstream RhoA and Cdc42 signaling [37]. Additionally,
MDA-9 was shown to play a key role in stabilizing integrin .beta.1
signaling complexes [38]. Hence, we wondered whether integrin
.beta.1 might also be involved in this MDA-9-TGF.beta.1 signaling
axis. The role of the extracellular matrix, comprised of integrins,
cannot be ignored when trying to understand tumor attributes such
as invasion [39]. In fact, integrins are key players that enhance
breast cancer invasion and metastasis [40, 41] and integrin .beta.1
plays a key role in metastatic progression of breast cancer
[42-44]. Hence we added an integrin .beta.1 blocking antibody to
SUM159 cells and assessed cytoskeletal rearrangement (FIG. 31A).
Addition of integrin .beta.1 blocking antibody caused a change in
cytoskeletal organization. Next, we added an integrin .beta.1
blocking antibody to T47D cells overexpressing MDA-9 and observed a
change in cytoskeletal organization (FIG. 3 IB). Our observations
and previous studies indicate that integrin .beta.1 might also be
involved in MDA-9 mediated regulation of the small GTPases RhoA and
Cdc42. This finding is consistent with published reports that show
that MDA-9 plays a key role in the assembly of integrin .beta.1
signaling complexes and that silencing the expression of MDA-9
impaired assembly/formation of several integrin .beta.1 signaling
complexes [38, 45]. Additionally, silencing the expression MDA-9
also resulted in inhibition of active integrin .beta.1 expression
(and downstream phosphorylation of ERK1/2) in breast cancer cells
[19]. Thus, the overall signaling mechanism mediated by MDA-9 in
breast cancer is illustrated in FIG. 26G.
[0697] In summary, our study has identified a novel role of MDA-9
in mediating EMT and enhancing invasive abilities in breast cancer
cells. Additionally, our findings provide preclinical evidence that
MDA-9 is an effective therapeutic target against breast cancer
including metastatic breast cancer.
[0698] Materials and Methods
[0699] Cell Lines and Cell Culture.
[0700] Human breast cancer cells T47D, ZR-75-1, SKBR3, BT-20,
MDA-MB-468 and MDA-MB-231 were purchased from the American Type
Culture Collection (ATCC) (Manassas, Va.) and cultured as
recommended by ATCC. T47D, ZR-75-1 and SKBR3 are epithelial cells
with low invasive and metastatic ability. BT-20 and MDA-MB-468 are
moderately invasive and metastatic. MDA-MB-231 is a highly invasive
and metastatic triple negative mesenchymal breast cancer cell line
[46, 47]. Human breast cancer cells SUM159PT (labeled SUM159
throughout) were purchased from Asterand, Inc. (Detroit, Mich.).
These cells were cultured in F-12 media supplemented with 5% fetal
bovine serum, 10 mM HEPES buffer, 5 .mu.g/ml insulin, 1 .mu.g/ml
hydrocortisone and 1% Penicillin/Streptomycin. SUM159 cells are
triple negative with strong abilities to invade and metastasize
[48]. ATCC authenticates these cell lines using short tandem repeat
analysis. All the cell lines were expanded and frozen immediately
after receipt. The cumulative culture length of the cells was less
than 6 months after recovery. Early passage cells were used for all
experiments. All the cell lines were frequently tested for
mycoplasma contamination using a mycoplasma detection kit from
Sigma. All of the cells were maintained at 37.degree. C. with 5%
CO.sub.2 in a humidified atmosphere.
[0701] Breast Cancer Tissue Microarray.
[0702] Tissue microarray comprised of breast cancer samples along
with metastatic and normal counterpart tissue samples was purchased
from Imgenex (San Diego, Calif.). Immunohistochemistry was
performed according to standard protocols. Briefly, tumor sections
were deparaffinized at 60.degree. C. for 1 hour, followed by
rehydration, and antigen retrieval using citrate buffer and
heating. Avidin and biotin blocking kits and Vectastain ABC complex
kits were obtained from Vector Laboratories (Burlingame, Calif.).
IHC-grade Prestige MDA-9 antibody was purchased from Sigma (St.
Louis, Mo.). Secondary antibodies were obtained from Jackson
Immunoresearch (West Grove, Pa.). The slides were counter-stained
using hematoxylin. Following staining, slides were dehydrated and
mounted using Vectashield mounting media (Vector Laboratories).
[0703] Preparation of Whole-Cell Lysates and Western Blotting
Analysis.
[0704] Cells were washed twice with ice-cold PBS and lysed in
1.times. Cell Lysis buffer (Cell Signaling Technology, Danvers,
Mass.) with protease and phosphatase inhibitors. The lysates were
kept on ice for 1 hour and centrifuged at 10,000 rpm for 30 minutes
at 4.degree. C. Protein concentration was measured using BioRad
protein assay reagent (Hercules, Calif.). Lysates corresponding to
equal amounts of protein were subjected to SDS-PAGE and transferred
on to PVDF membrane (0.2 .mu.m). The membranes were blocked with 5%
non-fat dried milk or bovine serum albumin (BSA) in TBST
(Tris-buffered saline with 0.1% Tween 20) and incubated with
primary antibodies overnight at 4.degree. C. The membranes were
then washed three times with TBST, incubated with respective
secondary antibodies for 1 hour at room temperature, washed three
times with TBST and then developed using ECL reagent (GE
Healthcare, UK). MDA-9/syntenin antibody was from Abnova (Taiwan),
.beta.-actin was from Sigma-Aldrich (St Louis, Mo.), EMT marker
antibodies were from Cell Signaling Technologies and TGF.beta.1 tag
antibody was from Origene (Rockville, Md.). EF1.alpha. was used as
a loading control and was obtained from EMD Millipore (Billerica,
Mass.).
[0705] RNA Extraction and qRT-PCR (Quantitative Real-Time PCR).
[0706] Cells were washed with PBS and then harvested using Qiazol.
RNAeasy kit (Qiagen, Germany) was used for RNA extraction according
to the manufacturer's protocol. RNA was converted to cDNA using the
cDNA synthesis kit (Applied Biosystems, Foster City, Calif.). The
PCR primers and probes were purchased from Applied Biosystems.
qRT-PCR was performed using the Applied Biosystems machine. The
gene expression .DELTA.C.sub.T values of mRNAs from each sample
were calculated by normalizing with GAPDH and relative
quantification values were plotted using GraphPad Prism.RTM..
[0707] Viruses, Plasmids and Reagents.
[0708] MDA-9 was silenced using Ad.5/3 shMDA-9 and overexpressed
using Ad.5/3 MDA-9. The construction of these viruses has been
described in detail previously [49]. For stably silencing the
expressing of MDA-9, lentiviruses targeting MDA-9 were purchased
from Sigma (St. Louis, Mo.). FLAG-tagged full length and PDZ-1
deleted constructs were obtained from TransOMIC (Huntsville, Ala.).
TGF.beta.1 construct was obtained from Origene. Human TGF.beta.1
protein was obtained from BioLegend (San Diego, Calif.) and the
TGF.beta.1 inhibitor (A83-01) was obtained from (Stemgent, San
Diego, Calif.).
[0709] Invasion Assay.
[0710] Invasion assays were performed using 24-well BioCoat
Matrigel.TM. invasion chambers (BD Biosciences, San Jose, Calif.)
in triplicates according to the manufacturer's instructions.
Briefly, the chambers were equilibrated to room temperature and
then rehydrated using warm serum-free cell culture media for 2
hours at 37.degree. C. Cells were seeded in serum-free media in the
upper chamber and serum-containing media was used as an attractant
in the lower chamber. Cells were allowed to invade through the
Matrigel.TM. overnight. The inserts were fixed with methanol,
non-invaded cells were wiped off using a cotton swab and the
inserts along with the invaded cells were stained with the
Diff-Quick staining kit. Cells that invaded were enumerated using
at least 8 fields per insert. Data is presented as
mean.+-.S.E.M.
[0711] Phalloidin Staining.
[0712] Cells were seeded in 4-well chambered glass slides and
allowed to attach overnight. The next day, the cells were fixed
with 4% paraformaldehyde for 1 hour, and permeabilized with 0.01%
Triton-X and 1% sodium citrate for 3 minutes on ice. The cells were
blocked with 1% bovine serum albumin for 30 minutes. The actin
cytoskeleton was stained using Alexa Fluor 488 Phalloidin (Life
Technologies, Carlsbad, Calif.) for 30 minutes. The wells were
detached from the glass slide and the slide was mounted using
mounting media containing DAPI (to label cell nuclei) (Vector
Laboratories).
[0713] 3D (Three-Dimensional) Culture.
[0714] Glass slides (eight-well chambered; Nunc, Rochester, N.Y.)
were coated with 3D Culture Matrix.TM. Basement Membrane Extract
Reduced Growth Factor (Phenol Red-free) (Trevigen, Gaithersburg,
Md.). A total of 5000 cells/well were seeded into the wells in
complete medium containing 2% 3D Matrix and the media was
replenished every 4 days. The slides were incubated at 37.degree.
C., humidified with 5% CO.sub.2 atmosphere [23, 50].
[0715] GTPase Activity Assay.
[0716] The RhoA G-LISA and Cdc42 G-LISA activation assay biochem
kits were obtained from Cytoskeleton Inc (Denver, Colo.) and GTPase
activity was measured according to the manufacturer's instructions.
Briefly, cells were cultured in serum-free media for 48 hours and
lysates were prepared using the appropriate G-LISA buffers. The
required number of G-LISA wells were rehydrated with ice-cold
water, and then lysates, lysis buffer (negative control) and
RhoA/Cdc42 control protein (positive control) were added to the
wells. The plate was incubated at 4.degree. C., followed by
incubation with antigen presenting buffer, primary antibody,
secondary antibody and then the HRP detection reagents. After
adding the HRP stop buffer, absorbance was measured at 490 nm.
[0717] Immunoprecipitation.
[0718] Immunoprecipitation was performed using the
immunoprecipitation kit with Dynabeads.RTM. Protein G (Life
Technologies) according to the manufacturer's instructions.
Briefly, MDA-9, TGF.beta.1, FLAG tag or IgG control antibody was
incubated with Dynabeads.RTM. and allowed to bind. Next, protein
lysates were incubated with the antigen bound Dynabeads.RTM..
Finally, the antibody-antigen complexes were eluted from the
Dynabeads.RTM., run on an SDS-PAGE gel and probed using the
appropriate antibody.
[0719] In Vivo Metastasis Study.
[0720] MDA-MB-231 control cells were stably transfected with the
luciferase expression plasmid pGL4.50 (Promega, Madison, Wis.)
specifically engineered to aid in vivo imaging. MDA-MB-231 control
cells stably expressing luciferase were selected using hygromycin.
MDA-9 stably silenced cells were similarly stably transfected with
the luciferase expressing plasmid. Both MDA-MB-231 control
luciferase and MDA-MB-231 shMDA-9 luciferase cells were stably
transfected with TGF.beta.1. 6-week old female athymic mice
(Charles River Laboratories, Wilmington, Mass.) were injected with
1.5.times.10.sup.6 cells of the above four cell lines through the
tail-vein. Five female athymic mice were injected per cell line.
The mice were monitored and luciferase expression was assessed by
bioluminescent imaging. The mice were sacrificed after 6 weeks. A
section of the lung was collected in DMEM/F-12 media supplemented
with Penicillin/Streptomycin. Tumor cells were harvested from the
lung metastasis by digesting the lung tissue using trypsin-EDTA.
Tumor cells were collected and cultured as per normal procedures.
Another section of the lungs was fixed in paraffin, sectioned and
H&E stained. Animals were maintained under the guidelines of
the National Institute of Health and under evaluation and approval
of the Institutional Animal Care and Use Committee (Virginia
Commonwealth University). Food and water were provided ad
libitum.
[0721] Statistical Analysis.
[0722] Results are presented as the mean.+-.S.E.M. for at least
three individual experiments. Statistical analyses were performed
using GraphPad Prism 5. Student's t-test was applied based on the
statistical mandates or suggestions of each analysis. p<0.05 was
considered statistically significant.
Example 4. Rationally Designed Small Molecule Inhibitors for
Preventing & Treating Cancer Metastasis
[0723] Disease complexity is a current barrier to progress in
metastasis drug development. Over 400+ cancer types with genotypic
heterogeneity. A 1.degree. Genetic lesions can lead to 1000s
2.degree. changes. Selective targeting of cancer-specific lesions
is problematic. Limited drug candidates that are efficacious
against advanced cancers.
[0724] Hundreds of drugs are targeting tumor cells, but none are
metastasis-specific. It seems like it should be easy to kill
metastatic tumor cells with the current knowledge of cancer
mechanisms, but it's not (FIG. 32). Cancer subtypes: 400+ and each
subtype exhibits molecular and biological heterogeneity. Evolution
through distinctive stages, culminating in lymph node and distant
metastasis. Lesional stages can evolve through genomic
rearrangement and reprogramming. Relapse and resistance are
common.
[0725] Opportunities for achieving durable responses against
metastasis include (1.) Single and Combinatorial therapies
targeting metastatic tumor cells (FIG. 33A) and (2.) Combinatorial
therapies aimed at the renegade organ, targeting the tumor and its
environment (FIG. 33B).
[0726] Metastatic tumors do not exist alone, they interact and
coexist with multiple cell types in the microenvironment.
Metastatic tumors represent complex (renegade) organ systems (FIG.
34).
[0727] There are, however, unique opportunities in cancer therapy,
including targeting multiple critical steps in metastasis such as
cancer cell attachment, invasion, and tumor angiogenesis. Therapies
aimed at multiple cell types and steps contributing to metastasis
have the greatest potential for success.
[0728] Therapies can target an essential gene mediating metastasis,
MDA-9/Syntenin/SDCBP, which is a pro-metastatic gene. Targeted
small molecule MDA-9 inhibitor (PDZ1i) can act to inhibit invasion,
attachment, and angiogenesis. Combined with other therapies, the
approach can be lethal to metastases. This represents a new
therapeutic approach "Anti-Invasion Therapy."
[0729] Tageting multiple steps in the metastatic cascade is
critical for effective therapy (FIG. 34).
[0730] We have harnessed a unique therapeutic opportunity targeting
MDA-9/Syntenin/SDCBP. MDA-9/Syntenin/SDCBP is a key gene regulating
multiple steps in metastasis. This gene provides a universal target
for therapy in diverse cancer settings. We have developed a first
in class small molecule inhibitor of MDA-9, PDZ1i. PDZ1i exhibits
good PK and exciting pre-clinical data. It is contemplated that
PDZ1i can also be used in combinatorial therapy, thereby creating
an anti-invasion therapy converted to cytotoxic therapy in multiple
cancers.
[0731] Bioinformatics confirms that mda-9 is a relevant gene in
multiple cancer indications, including melanoma, prostate cancer
and liver cancer (FIG. 35). MDA-9 expression correlates with cancer
progression in patient samples (samples taken from Melanoma,
Glioblastoma, Head and Neck Cancer, Urothelial cancer, Breast
Cancer, Uveal Melanoma, Gastric Cancer, Lung Adenocarcinoma,
Hepatocellular Carcinoma, Colorectal Cancer, Prostate Cancer,
Pancreatic Cancer, and Neuroblastoma) (FIG. 36). MDA-9 also
promotes tumor angiogenesis (FIG. 37). The crystal structure of
MDA-9/Syntenin shows two PDZ domains, PDZ1 and PDZ2 with an
interface between the PDZ domains. Applying FBDD and NMR applied to
purified PDZ1 and PDZ2 proteins identified PDZ1i (113B7) (FIG. 39).
PDZ1i efficiently binds with the PDZ1 domain of MDA-9 (Kd<10
.mu.M), shows low clearance from serum (T.sub.1/2>9 hrs), and
has 80% bioavailability when administered I.P. PDZ1i showed no
apparent toxicity in vivo in multiple pre-clinical animal models
and was non-toxic to both primary normal cells and cancer cells.
PDZ1i is an anti-invasive, anti-angiogenic small molecule that can
synergize with conventional therapies in primary tumor and
metastatic tumor cells. PDZ1i alters protein-protein interactions
thereby inhibiting MDA-9 signaling functions: blocking invasion,
attachment and angiogenesis (FIG. 40). PDZ1i "anti-invasion"
therapy can be successfully combined with "cytotoxic therapy" to
treat primary tumor and metastatic tumor cells (FIG. 41). FIG. 42
shows mechanisms of metastasis prevention through the use of PDZ1i.
PDZ1i can inhibit invasion of primary tumors including melanoma,
bladder cancer, prostate cancer, GBM, pancreatic, colon, etc. The
following are examples of combinatorial approaches to treating
cancer using anti-Invasive therapy cytotoxic therapy;
PDZ1i+Sorafineb to treat liver cancer; PDZ1i+Radiation to treat
glioblastoma; PDZ1i+Temozolomide to treat glioblastoma; PDZ1i+MCL-1
Inhibitor to treat prostate cancer, breast cancer, and melanoma.
PDZ1i alone or in combination prior to or after surgical removal of
cancer tissue to prevent secondary metastases.
Example 5. Effective Pharmacological In Vivo Inhibitors of Cancer
Invasion and Metastasis
[0732] Cancer is a progressive disease that can culminate in tumor
cell populations that have acquired the capacity to invade
surrounding tissue or enter the bloodstream, attach at distant
sites in the body and form metastases. If diagnosed early, recent
advances in diagnosis and therapy may permit management and even
cures for many solid tumors. However, cancer frequently becomes an
intractable disease when tumor cells migrate beyond their primary
site into adjoining tissue or to distant regions in the body.
Although extensively examined, leading to the identification of
many critical signal transduction pathways, the fundamental
genetic/epigenetic causes of cancer invasiveness and metastasis
remain complex and require further clarification. Defining the
pivotal genomic changes occurring in diverse cancer cells that
control invasion and metastasis holds significant potential for
developing rationally based anti-invasive and antimetastatic
therapies for these invariably fatal components of the cancerous
phenotype.
[0733] The central goal is to develop effective pharmacological in
vivo inhibitors of cancer invasion and metastasis. Our innovation
centers around a novel gene discovered by subtraction
hybridization, melanoma differentiation associated gene-9
(mda-9)/syntenin, that based on bioinformatics and direct
experimentation represents a key contributor to cancer cell
invasion and metastasis in multiple cancer types. Using innovative
fragment- and structure-based approaches with NMR, small molecule
inhibitors of the critical protein-interacting PDZ1 domain of the
MDA-9/Sytenin protein have been identified and shown to inhibit in
vivo tumor cell invasion, circulating tumor cell attachment and
metastasis formation.
[0734] PDZ inhibitory molecules can be refined to enhance potency,
selectivity and pharmacological properties. Mechanistic studies
will focus on precisely how these inhibitors prevent melanoma cell
adhesion and metastasis. The role of mda-9/syntenin in development
of hepatocellular carcinoma (HCC) will be determined as well as the
utility of this gene or protein as a therapeutic target for
inhibiting HCC pathogenesis. Experiments will investigate the
therapeutic potential of MDA-9/Syntenin inhibitors in HCC; define
precisely how MDA-9/Syntenin promotes hepatocarcinogenesis; and the
molecular basis of mda-9/syntenin overexpression in HCC.
[0735] The role of mda-9/syntenin in prostate cancer (PC)
development and progression will also be interrogated. Studies will
elucidate the biological significance and mechanism of over
expression of mda-9/syntenin in PC; the involvement of the
MDA-9/IGF-1R/STAT-3 axis in PC metastasis; and the therapeutic
efficacy of MDA-9/Syntenin inhibitors in PC pathogenesis.
[0736] This research will provide biologically active cancer
inhibitory MDA-9/Syntenin PDZ specific small molecules that can
obstruct tumor cell invasion and metastasis (both cell adhesion and
subsequent colonization in secondary sites in the body).
Additionally, by combining PDZ-inhibitors with other conventional
therapeutic agents, such as chemotherapy, radiation therapy and
immunotherapy, future opportunities for developing efficacious
combinatorial therapies for preventing cancer invasion and
metastasis, thereby enhancing patient survival, may be an
achievable goal.
[0737] Although the main cause of death from cancer is metastasis,
the majority of research has focused on comprehending tumor
development and progression at the primary tumor site (1). In
principle, there are two defined modes of tumor dissemination,
i.e., invasion and metastasis, which employ both common and unique
strategies to accomplish movement from a primary tumor,
colonization at a new site in the body and survival and expansion
at the new site (2,3). The multistep nature of tumor dissemination,
particularly metastasis, provides significant obstacles to
effective therapy (4,5-8). In these contexts, understanding the
molecular determinants, signaling pathways and biology of tumor
cell invasion and metastasis will be pivotal if one hopes to
develop efficacious therapies for these invariably fatal
consequences of cancer (5,6,9).
[0738] Overarching Problems and Barriers to Progress in the
Field.
[0739] A fundamental question is, "why is cancer dissemination
(invasion and metastasis) so difficult to treat?" Many factors
contribute to the failure of current therapies to impact on this
invariably fatal component of the carcinogenic process. Tumor cells
from the same and different organ sites are heterogeneous with
numerous genetic and epigenetic changes that are exacerbated as
cancers progress (7,8). Additionally, the sensitivity for detecting
metastatic tumors has current limitations (10-13) and therapeutic
approaches frequently encounter tumor-resistance mechanisms
engendering opposition to therapy-induced apoptosis and toxic
autophagy with enhanced tumor survival in suboptimal environments
(14-18). Localized invasion, which is the mode of pathogenesis in
glioblastoma multiforme (19-21), bladder cancer (22,23) and the
initial stages of melanoma (9,24), involves as a minimum partial
separation from the primary tumor, movement through tissue barrier
matrices and survival in contiguous tissues (2,3). In these
contexts, an invasive tumor is in fact an extension of the primary
tumor mass and progresses in a syncytial manner as it expands in
size. In this context, agents that provide effective therapies for
tumors depending on this mode of pathogenesis would benefit from
anti-invasive approaches (2). Metastasis is often viewed as a
temporal process, where different genes and signal transduction
pathway changes may regulate specific components of this process,
i.e., escape from the primary tumor and invasion of tumor cells
from a primary tumor mass into the circulation ("intravasation");
survival in the circulatory system; secondary attachment and
seeding in a new site in the body involving adherence to
endothelial cells and penetration through the basement membrane to
enter the tissue parenchyma ("extravasation"); and colonization and
development of the secondary tumors with a new blood supply
(metastases) (3,3,6,9,25). Metastasis has also been viewed as a
multistep process involving a series of gene and signal
transduction pathway changes referred to as: metastasis initiating
genes; metastasis progression genes; and metastasis virulence genes
(6, 9) (FIG. 42). A key would be to define common genomic changes
and pathways) that may mediate both invasion and metastasis in
multiple cancers, which are necessary for cancer cells but
dispensable for expression in normal cells, that could be targeted
by drugs to effectively inhibit specific stages in invasion and
metastasis (6,9,26). A primary objective is to develop small
molecule drugs to effectively inhibit cancer invasion and
metastasis.
[0740] MDA-9/Syntenin: novel pro-metastatic gene/protein with
potential as a therapeutic target. Using subtraction hybridization,
a novel temporally expressed gene was cloned, melanoma
differentiation associated gene-9/syntenin (mda-9/syntenin) from
human melanoma cells induced to undergo terminal differentiation
(27,28). MDA-9/Syntenin is a protein, which contains two PDZ
domains, PDZ1 (aa, 110-193) and PDZ2 (aa, 194-274), that have
potential to interact with a plethora of proteins, many of which
are relevant to the cancer phenotype (9,29,30). Studies in melanoma
indicate that MDA-9/Syntenin functions as an adapter protein
interacting with multiple partners and provides a central role in
regulating cell-cell and cell-matrix interactions (9,29-33).
MDA-9/Syntenin transduces signals from the cell-surface to the
cell's interior through its physical communications with a broad
spectrum of additional proteins (9,29,30). Recent studies provide
compelling evidence that MDA-9/Syntenin plays a central role in
cancer cell motility, invasion and metastasis (9,21,22,29-39), and
functions as a positive regulator of tumor angiogenesis (40). In
the context of human melanoma, MDA-9/Syntenin serves as a positive
regulator of progression and metastasis through interactions with
c-Src and promotes the formation of an active FAK/c-Src signaling
complex leading to NF-.kappa.B and matrix metalloproteinase (MMP)
activation (FIG. 43) (36-39). Additionally, through interaction
with the extracellular matrix, MDA-9/Syntenin induces a cascade of
molecular/biochemical changes culminating in tumor angiogenesis
(40).
[0741] MDA-9/Syntenin through c-Src and FAK promotes activation by
phosphorylation of Akt inducing HIF-1.alpha. resulting in the
transcription of insulin growth factor-binding protein-2 (IGFBP-2)
which upon secretion promotes angiogenesis and also induces
endothelial cells to produce and secrete VEGF-A augmenting further
tumor angiogenesis (FIG. 44) (40). With respect to the model
delineating multiple types of metastasis regulating genes as shown
in FIG. 42, evidence is available that MDA-9/Syntenin displays
properties of all three components in this multistep and
multifactor process, i.e., metastasis initiation and metastasis
progression (motility, invasion, angiogenesis, intravasation) and
metastasis progression and metastasis virulence (survival in the
circulation, capillary adhesion, extravasation)
(9,21,22,29,30,33,37-41). Moreover, as emphasized ectopic
expression of MDA-9/Syntenin in a normal immortal melanocyte
promotes all of the properties necessary for induction of
metastasis in an experimental metastasis model following injection
into the bloodstream (39).
[0742] MDA-9/Syntenin, Syndecan-Binding Protein (SDCBP), is
Upregulated in Multiple Cancers:
[0743] There is now significant bioinformatics evidence that
MDA-9/Syntenin (syndecan binding protein; SDCBP) is a relevant gene
in multiple cancer indications. Our comprehensive analyses of
various genome-wide expression datasets (such as those from The
Cancer Genome Atlas and NCBI's Gene Expression Omnibus) indicate
that MDA-9/Syntenin is commonly upregulated in numerous cancer
types. The positive correlation between MDA-9/Syntenin expression
and tumor grade is readily observed in glioma, upon analyses of
both the TCGA (Glioblastoma Multiforme plus Lower Grade Glioma,
Illumina HiSeq 2000 platform) and GSE4290 (from NCBI-GEO; Asymetrix
U133 Plus 2 platform) datasets (21). Other tumors exhibiting
elevated MDA-9/Syntenin expression (relative to normal tissues) are
kidney renal papillary carcinoma (TCGA; Alumina HiSeq 2000),
melanoma (GSE3189; Affymetrix U133A),prostate adenocarcinoma (TCGA;
Alumina HiSeq 2000), and hepatocellular carcinoma (TCGA; Alumina
HiSeq 2000) (FIG. 35).
[0744] Novel Chemical Biological Approach Identifies MDA-9/Syntenin
PDZ Domain Inhibitors:
[0745] Targeting the interaction between MDA-9/syntenin's PDZ1
domain and its targets through novel small molecular inhibitors and
developing suitable pharmacologically acceptable drugs using
innovative fragment- and structure-based approaches holds
significant potential for producing effective therapeutics for
inhibiting metastatic disease progression in a broad-spectrum of
human cancers. Using an innovative modular approach, a series of
inhibitors targeting the PDZ1 domain and the interface between the
domains were derived These initial chemical probes were used to
further our understanding of the role of MDA-9/Syntenin in
mediating invasive behavior of tumors using animal models.
Additionally, we intend to obtain the structural determinants that
are the basis of the binding of these molecules using solution NMR
spectroscopy and to use the resulting structures to refine the
molecules for potency, selectivity and also pharmacological
properties as needed. This chemical biology approach will likely
provide new insights into metastasis mediated by mda-9/syntenin in
melanoma, Aver, and prostate cancer.
[0746] Development of MDA-9/Syntenin Targeted Small Molecules:
[0747] The prime objective is to develop small molecules that can
selectively inhibit the interactions between the PDZ1 domain of
MDA-9/Syntenin with critical interacting proteins. Molecules
capable of specifically targeting the PDZ1 and interdomain (e.g.,
interface region) of MDA-9/Syntenin, such as 113B7 (PDZ1in), are
effective inhibitors of the subsequent biological properties
induced by MDA-9/Syntenin. Existing PDZ1 inhibitory molecules will
be refined in an effort to enhance potency, selectivity and
pharmacological properties. The biological and molecular properties
of specific inhibitors, including effects on normal cell survival,
cancer cell invasion, protein-protein interactions, retention of
tumor cells in the lungs and generation of metastatic lesions in
vivo, will be ascertained. Mechanistic studies will define the
pathways and critical genes associated with enhanced invasion,
attachment and metastasis that are manipulated by overexpressing or
inhibiting MDA-9/Syntenin expression in human melanoma cells. This
combination of medicinal chemistry and preliminary biological
characterization (to define appropriate functional and mechanism of
action studies in the context of metastatic B16 mouse and human
MeWo and C8161 melanoma cells will provide essential insights for
studies in HCC and prostate carcinoma. Further refinement and
testing of clinical efficacy will also be performed.
[0748] Targeting MDA-9/Syntenin to Treat Hepatocellular
Carcinoma:
[0749] Although the overall incidence and mortality of the majority
of cancers is decreasing HCC is one of the few cancers where the
incidence and mortality is alarmingly increasing for several
decades (49,50). HCC, detected in early stages, might be remedied
by surgical resection, radio-embolization and liver transplantation
(49,51). However, most patients present at an advanced stage with
intra-hepatic and distant metastasis, which is not malleable by
standard modalities of treatment. The only FDA-approved drug,
sorafenib, for non-resectable HCC provides a survival advantage of
only 2.8 months (52,53). Understanding the molecular mechanism of
hepatocarcinogenesis and developing targeted therapies are thus
mandatory to effectively counteract this fatal disease. 113B7
(PDZ1in) will be evaluated as a therapeutic targeted towards
inhibition of invasion and metastasis. We propose to combine PDZ1in
with sorafenib for the following rationale: (1) Even though 113B7
(PDZ1in) is a potent inhibitor of invasion, it may not affect
proliferation of human HCC cells. PDZ1in may benefit from being
combined with a drug that inhibits proliferation of cancer cells
and induces apoptosis and sorafenib provides those functions
(54,55). (2) Sorafenib is a relatively non-specific kinase
inhibitor. However, it targets Raf-1 and VEGFR, and downstream
signaling from these kinases has been shown to be active in HCC
(56-58). We anticipate that the anti-proliferative and
anti-angiogenic functions of sorafenib will complement
anti-invasive and antimetastatic function of 113B7 providing a
greater synergistic effect. (3) Since sorafenib is already used in
the clinic, if our proposed strategy is proven successful, it will
allow fast-track translation into the clinic to provide immediate
benefits to scores of HCC patients.
[0750] Targeting MDA-9/Syntenin to Treat Prostate Cancer:
[0751] Although there have been improvements in radiotherapy,
chemotherapy and hormone therapy, this has not translated into
overall long-term survival benefits in patients, particularly in
the context of advanced prostate cancer (PC) (metastatic stage).
Consequently, defining the crucial molecules controlling
progression of adenocarcinoma of the prostate and defining
rationally targeted and more efficacious therapies based on precise
mechanisms of pathogenesis are mandatory for developing treatments
that are potentially curative. We focus on critically evaluating
the role of mda-9/syntenin in PC etiology and progression, with
particular emphasis on the invasive phenotype (using the Hi-Myc
transgenicmouse model) and metastasis. This will test the
prevailing hypothesis underlying the mechanism of action of
MDA-9/Syntenin that by physically interacting with specific subsets
of proteins in different cancer cells, MDA-9/Syntenin regulates
crucial signaling pathways that enable cancer cell invasion and
metastasis. Our focus will be on PC with potential to identify
unique small molecules that through disruption of MDA-9/Syntenin
interactions with relevant partners will impact on final cancer
phenotypes in PC cells, with specific attention on invasion and
metastasis. Our emphasis in PC will be on defining the outcome of
interactions between MDA-9/Syntenin and insulin like growth factor
receptor-1 (IGF-R1) and ensuing STAT-3 signaling. The impact of
disrupting this complex with existing PDZ1 inhibitor 113B7 (PDZ1in)
will be further investigated. We will test further potential
MDA-9/Syntenin PDZ1in in suppressing tumor growth, invasion and
progression to metastasis.
[0752] Based on genomic relevance of MDA-9/Syntenin in multiple
types of cancer (TCGA, Oncomine, GEO) and experimental evidences
indicating a pivotal role of this scaffold protein in promoting
tumor cell motility, invasion, metastasis and angiogenesis
(9,21,22,29-40), targeting MDA-9/Syntenin or its downstream
regulated molecules may provide a means of simultaneously blocking
invasion and metastasis. This antiinvasive/anti-metastatic activity
of blocking MDA-9/Syntenin could occur by directly inhibiting tumor
cell transformed properties (autonomous) and indirectly by blocking
angiogenesis (nonautonomous). This multi-pronged attack on a
pivotal gene that directly impacts on cancer aggressiveness
provides significant potential to develop effective anti-invasive
and anti-metastatic drugs, which is a primary objective of cancer
treatment.
[0753] We have shown that targeting MDA-9/Syntenin protein through
inhibiting interaction of its PDZ1 domain with critical effector
proteins provides a viable approach for developing small molecules
that can affect cancer progression both in vitro and in vivo in
animal models. These results could translate into a paradigm shift
of how cancer invasion and metastasis are treated. The approach of
using a single inhibitor that combines anti-invasive,
anti-metastatic and anti-angiogenic activity in the same molecule
in conjunction with therapies that inhibit cancer cell growth and
survival, is innovative and provides a new Multi-Modality
Gene-Specific (MMGS) cancer therapeutic approach to inhibit and
potentially prevent cancer pathogenesis mediated through cancer
invasion and metastasis.
[0754] Elevated MDA-9/Syntenin Expression Correlates with Disease
Progression in Multiple Cancers:
[0755] Through bioinformatics interrogating various cancer data
bases we confirm a correlation between elevated expression of
mda-9/syntenin and advanced stages of multiple cancers, including
melanoma, HCC and PC (FIG. 35). A direct relationship between
MDA-9/Syntenin expression and progression of human melanoma has
been documented using tissue sections representing normal and
various stages of superficial spreading and uveal melanoma (35,39).
In the context of urothelial cell carcinomas, a significantly
higher expression of MDA-9/Syntenin was observed in 64% (28 of 44)
of primary UCC tumors and an association was evident with stage,
grade and invasion status (22). In this study, a direct physical
interaction and colocalization of MDA-9/Syntenin and EGFR was
evident in UCC cell lines and primary tumors. These studies support
MDA-9/Syntenin as an attractive target for developing detection,
monitoring and therapeutic strategies for managing UCC (22). In the
context of astrocytoma development and progression to GBM, a
correlation also exists between expression of mda-9/syntenin and
disease progression. In GBM patients, there is a direct
relationship between elevated mda-9/syntenin expression and poor
patient prognosis with decreased survival. Using both gain and loss
of function studies, mda-9/syntenin was shown to positively
regulate astrocytoma motility and invasion, in vitro and in vivo
(21). The studies in UCC and GBM are particularly relevant in the
context of tumor cell invasion, which is a primary mode of
pathogenicity of these cancers. A correlation between migration and
metastatic human breast and gastric cancer cell lines has also been
reported (41). These studies demonstrate that mda-9/syntenin is a
highly relevant gene in progression of multiple cancers, including
regulation of processes including invasion and metastasis.
[0756] MDA-9/Syntenin Expression Directly Correlates with Tumor
Progression in Patient Samples and Invasive and Metastatic
Phenotypes in Cancer Cells:
[0757] Although bioinformatics data is important in providing
potential leads and tentative associations between specific gene
expression changes and cancer phenotypes, experimental
documentation of these changes in primary tumors, cell culture and
animal models is compulsory to verify these relationships. Detailed
studies in normal melanocytes and various stages of melanoma
(including metastatic cells) (35,39), normal bladder cells and
urothelial cell carcinoma (UCC) (22), normal hepatocytes and HCC,
normal prostate and PC and astrocytes, low grade astrocytomas and
GBM (21) demonstrate a direct correlation between MDA-9/Syntenin
expression and tumor histological grade. Using genetic gain and
loss of function others (31,32,34,35) and we (21,22,36-40)
confirmed a direct relationship between MDA-9/Syntenin levels and
in vitro and in vivo (animal models) expression of transformed
properties, including tumor cell dissemination (motility, invasion,
metastases). In the context of UCC and GBM, inhibiting
mda-9/syntenin suppressed invasion, whereas overexpression of
mda-9/syntenin in normal or less aggressive tumor cells facilitated
motility and invasion (21,22). Direct in vivo injection into mouse
brains of genetically modified grade III astrocytoma (elevated
MDA-9/Syntenin) and GBM (inhibition of MDA-9/Syntenin) confirm a
direct relationship between expression of mda-9/syntenin and tumor
spread, lethality (21). In metastatic melanoma, inhibiting
mda-9/syntenin expression blocks tumor cell retention in lungs and
metastasis. Overall, these studies highlight the importance of
MDA-9/Syntenin as a positive regulator of cancer dissemination and
confirm that inhibiting expression impedes tumor cell invasion and
metastasis.
[0758] Application of FBDD, NMR and structure-based design
identifies MDA-9/Syntenin specific small molecule inhibitors:
Recent years have witnessed a growing interest in targeting PDZ
domains with relatively high success using small peptides, natural
products and small molecules. These initial accomplishments are
quite exciting and suggest that the PDZ domains are "draggable"
(S9-6S). There are over ISO PDZ domains in the human genome
discovered thus far (66,67). However, while these proteins share
similar global folds, their binding surfaces are quite distinct.
For this reason, developing small-molecule inhibitors targeting
specific PDZ/target interactions is an attainable objective. Here
we propose to use a combination of structure- and NMR-guided Drug
Discovery approaches (68-70) to derive focused libraries of
MDA-9/Syntenin PDZ targeting ligands. Our preliminary data
documents that these strategies have enabled the identification and
initial optimization of small molecules capable of antagonizing
MDA-9/Syntenin in vitro and in cells targeting its PDZ1 domain.
[0759] Biological efficacy of small molecules targeting the PDZ1
domain of MDA-9/Syntenin in suppressing cancer
dissemination-invasion and metastasis: As briefly described above,
using innovative approaches a structurally distinct small molecule
inhibitor of MDA-9/Syntenin PDZ1 domain has been produced, 113B7
(interacts with PDZ1 and the interdomain (e.g., interface region)
of MDA-9/Syntenin; PDZ1in). Bioinformatics suggest that
MDA-9/Syntenin expression is elevated in a large panel of human
tumors, which was confirmed by Western blotting (39). Based on
these findings, we determined the effect of 113B7 (PDZ1in) on
invasion of multiple cancer cell types including melanoma,
prostate, HCC, GBM, pancreatic and breast (FIG. 46). 113B7
inhibited invasion in a dose-dependent manner in various cancer
cell types, including melanoma, prostate and HCC.
[0760] To confirm specificity of this effect, we genetically
modified normal immortal human melanocytes (FM516), normal human
prostate epithelial cells (RWPE) and normal human mesenchymal
pancreatic cells to stably express elevated levels of
MDA-9/Syntenin. These MDA-9/Syntenin over-expressing cells
displayed elevated levels of invasion, which was inhibited when
treated with 113B7 (FIG. 47). In metastatic human melanoma and PC
cells, respectively, 113B7 effectively reduced retention time of
tumor cells in the lungs, possibly by preventing attachment, and
113B7 inhibited development of metastases in animal models (FIG.
48). A direct anti-invasive effect of 113B7 in the context of
Hi-Myc mice was also documented (FIG. 48). Additionally, the
combination of 113B7 and sorafenib synergistically inhibited HCC
growth in nude mice (FIG. 48). These experiments show the invasive
suppressing and anti-metastatic properties of PDZ1in (113B7),
suggesting the potential of this molecule to serve as scaffolds for
developing novel therapeutics against a broad spectrum of human
cancers.
[0761] Novel Molecular Mechanism of Hepatocarcinogenesis Involving
Interaction of MDA-9/Syntenin, AEG-1 and EGFR on the Cell Membrane
of Human HCC Cells:
[0762] Overexpression of the oncogene Astrocyte elevated gene-1
(AEG-1), also known as metadherin (MTDH), has been documented in
all cancers studied so far, including HCC (71-73). AEG-1 expression
level correlates with poor disease prognosis, decreased overall
survival and disease recurrence (71-73). AEG-1 positively regulates
all hallmarks of cancer but its more pronounced effect is observed
on invasion, metastasis and angiogenesis (71-73). The cell membrane
located AEG-1 has been ascribed to promote metastasis of breast
cancer cells to the lungs (74). Overexpression of EGFR has been
documented in HCC patients and contributes to invasion and
metastasis by activating c-Src and c-Met (75,76). We now document a
novel interaction among MDA-9/Syntenin, AEG-1 and EGFR on the cell
membrane of human HCC cells that might contribute to aggressive
progression of hepatocarcinogenesis (FIGS. 49 and 50). Detailed
analysis of the consequence of these interactions will provide
novel insights into regulation of growth factor signaling, as well
as mechanism of regulation of invasion and metastasis by
MDA-9/Syntenin and AEG-1. These studies complement mechanistic
studies in melanoma.
[0763] Unique link between MD A-9/Syntenin/IGF-R1/STAT3 in prostate
cancer development and progression: Progression of prostate cancer
(PC) to hormone resistance and metastasis are major problems in
clinical management, which negatively impact survival. Accordingly,
identifying the molecular changes that lead to invasion and distant
metastases is critical for developing improved approaches to
delimit these processes and enhance patient outcome. Based on
genomic databases and pre-clinical studies using patient samples,
we have demonstrated that MDA-9/Syntenin expression is associated
with high histological grade and advanced cancer stage. At a
molecular level, MDA-9/Syntenin activates the transcription factor
STAT3, whose aberrant activation is associated with advanced stages
of PC (79,80) through interacting with Insulin Growth Factor-1
Receptor (IGF-1R) and may contribute to metastatic progression (81)
(FIGS. 49 and 50). Furthermore, a unique link involving this
signaling cascade (MDA-9/Syntenin/IGF-R1/STAT3) with metastatic
growth is established through experimental evidences indicating
that inhibition of metastasis is achievable by selectively
disrupting the interaction between MDA-9/Syntenin and IGF-1R (FIG.
50).
[0764] mda-9/syntenin was cloned in 1995 (26,27) and has been shown
through recent bioinformatics and direct experimentation to be a
critical component in cancer progression, serving as a direct
regulator of cancer cell invasion and metastasis (21,22,29-41). The
discovery that MDA-9/Syntenin is a major player in defining tumor
dissemination in multiple cancer contexts represents an innovative
observation that we are exploiting to develop novel anti-invasive
and anti-metastatic therapies. Using state-of-the-art chemical
approaches that employ fragment-based drug-discovery (FBDD)
approaches in combination with NMR unique small molecules that can
interface with the PDZ1 domain (PDZ1in; 113B7) of MDA-9/Syntenin
have been identified. These inhibitors have been shown to have
remarkable activity in suppressing invasion of a broad spectrum of
cancer cells that overexpress MDA-9/Syntenin in vitro (FIGS. 46 and
47), and invasion and metastasis in vivo in animal model systems
(FIG. 48).
[0765] Protein-protein interaction databases suggest that the PDZ1
motif of MDA-9/Syntenin can interact with a spectrum of proteins
(66),including a predicted 151 of 250 proteins associated with the
cancerous phenotype (67). Using the creative approaches, molecules
unique to the interface of the PDZ1 domain of MDA-9/Syntenin have
been generated confirming the power of the FBDD approach plus NMR
for identifying small molecule inhibitors specific to this
molecule. In the context of HCC, an important interaction at the
cell surface of HCC cells between MDA-9/Syntenin, an oncogene AEG-1
(19,20,72,73,82), and EGFR has been demonstrated and characterized
(83) (FIGS. 49 and 50) that will provide novel insights into
hepatocarcinogenesis. Treatment with 113B7 in combination with
sorafenib was also shown to inhibit HCC tumor growth in athymic
nude mice, supporting the potential application of this
anti-invasive and anti-metastatic agent in combination with other
therapeutic agents in potentially treating liver cancer (FIG.
48).
[0766] An innovative discovery indicates that MDA-9/Syntenin can
interact with IGF-R1 and this interaction is pivotal for activating
STAT3 (FIGS. 49, 50, and 51) and this interaction is pivotal in PC
development and progression. This discovery suggests potential ways
of using PDZ inhibitors to treat PC.
[0767] The present program benefited from state-of-art approaches,
FBDD and NMR guided structure based design, to identify small
molecules that selectively bind to the PDZ1 domain of
MDA-9/Syntenin, 113B7(PDZ1in) (FIG. 45). Once initial hits are
verified (non-toxic to normal cells, suppress invasion in cancer
cells and block specific protein-protein interactions), new
variants will be prepared using medicinal chemistry approaches.
These new small molecules will undergo a series of evaluations to
define appropriate molecules to ultimately be tested for in vivo
activity, using lung retention assays and metastasis development.
The overall strategy to be used will involve: 1) initial evaluation
for a lack of growth inhibitory or toxic properties in normal
immortal melanocytes, hepatocytes and prostate epithelial cells
using MTT assays; 2) screening of non-toxic compounds for effects
on proliferation (MTT assays) and invasion (96-well invasion
assays) of human melanoma, HCC and PC cells; 3) confirming the
effect of biologically active potential lead compounds as
inhibitors of specific protein-protein interactions between the
PDZ1 domain and defined interacting molecules (including src, EGFR
and IGF-R1) using co-immunoprecipitation and colocalization
analyses (FIGS. 49 and 50); and 4) define safety and efficacy in
melanoma, HCC and PC animal models when injected IP and IV. Since
small molecule development is an iterative process, compounds
showing desired activity will then be used as scaffolds for
developing new small molecules by appropriate chemical biological
approaches. Compounds will be tested in the context of melanoma,
HCC, PC, and for defining efficacy against specific metastases
(including those induced by melanoma, HCC and PC).
[0768] Preliminary results include the design and characterization
of a first series of compounds targeting PDZ1 using a combination
of FBDD techniques guided by structure and NMR-based design.
Fragment hits were found that bind to the PDZ1 domain (F1 in FIG.
45) and at the interface between the domains (F2 in FIG. 45). An
initial F1 PDZ1 fragment hit (FIG. 45) was selected among these
based on affinity by NMR and ITC (Kds ranging between 30 and 300
.mu.M for about 30 compounds analogues of this class tested) and by
the fact that the fragment hit appears non-cytotoxic (<50 .mu.M)
to both primary immortal melanocytes and aggressive melanoma cells.
Guided by NMR structural studies and docking, have generated novel
and effective bi-dentate PDZ1 inhibitory compounds spanning both
the PDZ1 and an interface pocket (FIG. 45). The bi-dentate compound
of this series represented by 113B7 has been characterized using
NMR techniques and it displays dissociation constant in the low
micromolar range according to NMR-titration binding assays. PK
studies with 113B7 indicate that the compound is stable and
long-lived (T1/2>9 hr) with nearly 80% bioavailability when
administered I.P. Interestingly, this compound spans both the first
PDZ domain and the interface between the two domains
(experimentally verified by NMR), but it does not appreciably bind
to the second PDZ domain, further corroborating our central
hypothesis that it possible to obtain high affinity, selective PDZ
antagonists. The effect of 113B7 on invasion was tested using a
panel of human cancer cells derived from different sites, including
melanoma, pancreas, prostate, breast, liver (HCC) and brain (GBM)
(FIGS. 46 and 47). These cancer cell types were chosen because they
display a high basal level of mda-9/syntenin expression at both
mRNA and protein levels compared with corresponding primary normal
cell counterparts.
[0769] Pretreatment with 113B7 (PDZ1in) significantly inhibited the
invasion of all of these cancer cells (FIG. 46) and invasion and
metastasis in vivo (FIG. 48). Similar to the F1 fragment, the
bi-dentate compound did not show any significant cytotoxicity, when
tested up to 20 .mu.M.
[0770] Another goal is to comprehend the mechanism by which
mda-9/syntenin expression is upregulated in human HCC, unravel the
molecular mechanism by which protein-protein interactions mediate
pro-invasive and pro-metastatic functions of MDA-9/Syntenin, and
evaluate a novel MDA-9/Syntenin inhibitor as a potential
therapeutic for HCC. Thus, the overall approach is to understand
the fundamental molecular mechanism regulating development and
progression of HCC and exploit the garnered knowledge to develop
novel therapeutics. The analysis of molecular mechanism of
mda-9/syntenin upregulation might facilitate employment of
strategies to block mda-9/syntenin expression as potential HCC
therapeutics. Mapping of the protein-protein interaction domains
between MDA-9/Syntenin and its interacting partners will pave the
way for developing small molecule or peptidomimetics interrupting
these interactions thereby inhibiting HCC. Finally, the evaluation
of 113B7 next generation inhibitors, with sorafenib combinatorial
treatment might facilitate immediate implementation of this
strategy to treat HCC patients. Thus, our approach provides
immediate translational significance and will accrue important
insights to develop future targeted therapies.
[0771] Another goal is to comprehend the molecular mechanism by
which mda-9/syntenin expression is upregulated in human PC, define
the role of mda-9/syntenin in regulating PC stem/progenitor cell
phenotypes, evaluate the MDA-9/Syntenin/IGF-1R/STAT-3 axis in PC
metastasis and investigate novel MDA-9/Syntenin PDZ-inhibitors as
potential modulators of PC pathogenesis. We will interrogate
transcriptional regulation of mda-9/syntenin in PC cells as
modulators of enhanced expression using appropriate molecular
biological techniques. The role of mda-9/syntenin in maintenance of
sternness, tumor evolution, angiogenesis and metastasis will be
evaluated using both in vitro 3-dimensional culture systems and in
vivo nude mouse models. We will explore the relevance of the
observation that MDA-9/Syntenin physically interacts with insulin
growth factor-1 receptor (IGF-R1) (FIG. 49), which activates
STAT-3. Deletion analysis will be used to define the regions of
MDA-9/Syntenin and IGF-R1 that interact and their functions and the
downstream signaling pathways affected by these interactions.
Studies will focus on the role of specific MDA-9/Syntenin PDZ
inhibitors in modulating PC invasion using the Hi-myc mouse (FIG.
48). This inhibitor could also be evaluated in combination with
cytotoxic drugs or immune mediators to define further enhancement
in clinical activity.
[0772] Based on primary screening (invasion assays) using a series
of compounds developed using a combination of FBDD techniques
guided by in silico docking and NMR-based design a small molecule
PDZ inhibitor (PDZ1in) was selected for further validation. Initial
validation of PDZ1in compound inhibited the invasive properties of
cancer cells from multiple anatomic origins (FIGS. 46 and 47).
Consistent with in vitro studies, in in vivo models the compounds
demonstrated anti-metastatic efficacy in invasion after inhibitor
treatment (FIGS. 46 and 47) and in immunocompetent C57BL/6 mice
(FIG. 48A).
[0773] In addition, the anti-tumorigenic (through inhibition of
angiogenesis) and anti-invasive role of PDZ1in was also confirmed
in HCC derived xenograft model (FIG. 48B) and spontaneous prostate
cancer transgenic model, respectively (FIG. 48C). Further studies
showing that MDA-9/Syntenin as an adaptor protein can also interact
with multiple protein(s), e.g., AEG-1 (in HCC), IGF-1R (FIG. 49)
(in PC) and affect tumor progression, which was blocked by PDZ1in
(FIG. 50). Novel observations in PC identify an MDA-9/IGF-R1/STAT3
link in PC development and progression (FIG. 51). Pharmacokinetics
studies were performed to obtain insights about the druggable
characteristics demonstrating that PDZ1in is stable (T1/2>9 hr)
and bio-available (.about.80%) (injected I.P. or I.V.).
Example 6. Pharmacological Tools to Inhibit PDZ Domains of
MDA-9
[0774] Recent observations delineate a pivotal role of
MDA-9/Syntenin and its PDZ domains (1) in melanoma
progression/invasion/metastasis (2-5) (FIG. 44). Intriguingly, this
gene is overexpressed in several additional cancers (6, 7) and it
has been hypothesized as being potentially associated with tumor
evolution and metastatic disease progression in multiple human
neoplasms including melanoma, glioblastoma, liver, bladder, head
and neck, pancreatic and prostate, thereby establishing
MDA-9/Syntenin as a genuine potential global target for developing
cancer therapeutics blocking tumor invasion and metastasis,
mda-9/syntenin was cloned and shown to be an interaction hub
regulating through its PDZ domains (8-10) (FIG. 44), multiple
signal transduction pathways, including Src activation, FAK.
phosphorylation, p38 activation and JNK activation, all of which
are involved in tumorprogression, invasion and metastasis.
Additionally, mda-9/syntenin induces enhanced expression of a
number of secreted proteins that contribute to the cancer
phenotype. Based on the considerations described above, targeting
the interaction between MDA-9/Syntenin's PDZ1 domain and its
targets through novel small molecular inhibitors and developing
suitable pharmacologically acceptable drugs using innovative
fragment- and structure-based approaches holds significant
potential for producing effective therapeutics for inhibiting
metastatic disease progression in a broad-spectrum of human
cancers. Using a modular approach, we have derived inhibitors
targeting PDZ1 and the interface between the domains,
MDA-9/Syntenin. We intend to use these initial chemical probes to
further our understanding of the role of MDA-9/Syntenin in the
invasive and metastatic behavior of tumors using animal models.
Furthermore, we intend to obtain the structural determinants as the
basis of the binding of these molecules using solution NMR
spectroscopy and to use the resulting structures to refine the
molecules for potency, selectivity and also pharmacological
properties as needed. In addition to evaluating these new small
molecules in metastatic melanoma it will further be investigated in
hepatocellular carcinoma (HCC), and prostate cancer. Our studies
will provide new insights into metastasis mediated by a
pro-metastatic gene, mda-9/syntenin in a variety of human cancers.
As indicated, lead compounds will be evaluated in a variety of
models and if successful our studies will provide critical
validation. Based on these exciting findings and solid foundations,
we propose the following.
[0775] To Develop Innovative Pharmacological Tools Targeting PDZ
Domains of MDA-9/Syntenin.
[0776] Our chemical biology approach consists of parallel
optimizations/evaluation including performing structural studies
and structure-guided SAR on our initial PDZ inhibitor (PDZin)
compounds in complex with MDA-9/Syntenin to iteratively optimize
the compounds not only for affinity, but also for their
pharmacological properties (ADME-Tox), to increase the likelihood
that these resulting compounds will have cellular and in vivo
efficacy (Aim 2). Three parallel strategies are proposed in an
attempt to obtain specific ligands for the first PDZ domain of
MDA-9/syntenin.
[0777] Mechanistically Evaluate Current MDA-9/Syntenin PDZ
Inhibitors (PDZin) in Preventing Melanoma Cell Adhesion, Invasion
and Metastasis, and Identify/Characterize the Next Generation of
PDZin.
[0778] The biological and molecular properties of specific PDZ
inhibitors (PDZin), including effects on normal cell survival,
cancer cell invasion, protein-protein interactions, retention of
tumor cells in the lungs and generation of metastatic lesions in
vivo, will be performed. Mechanistic studies will focus on defining
the pathways and critical genes associated with enhancing invasion,
attachment and metastasis that are manipulated by overexpressing or
inhibiting MDA-9/Syntenin expression in human melanoma cells. This
combination of medicinal chemistry and preliminary biological
characterization, to define appropriate small molecules and
mechanistic studies in the context of metastatic B16 mouse and
human MeWo and C8161 melanoma cells, will provide necessary
insights for further studies, particularly in vivo experiments,
proposed in HCC and prostate carcinoma.
[0779] Metastasis: the ultimate challenge for effective cancer
therapy: Metastasis, is a multistep process involving migration of
cancer cells from their origin in a primary tumor to colonization
at distant sites, thereby posing significant challenges to drug
discovery (11). Although 90% of cancer-associated mortality is due
to metastasis and this process has been extensively studied, it
remains one of the most enigmatic aspects of the disease and
mandates an in-depth understanding of the sequence of events
orchestrating the metastatic cascade (11, 12). From a therapeutic
standpoint, elucidating the mechanisms leading to successful
physical translocation and colonization of tumor cells from primary
sites to loco-regional or distant sites holds promise for
developing effective therapeutics for preventing and treating
metastases (13-16). In principle, effective antimetastatic drugs
could be developed that target intravasation: initial detachment
and release from the primary tumor followed by mobilization through
the extracellular matrix of surrounding tissues, and survival in
the bloodstream; and/or extravasation at secondary sites, followed
by production of new blood vessels (angiogenesis) and
survival/growth of micro-metastatic lesions (11, 13, 15). The
global process is extremely complex involving interplay of multiple
signaling pathways, including sequential alterations of genetic and
epigenetic components. Consequently, elucidating the molecular
targets involved in tumor metastasis is mandatory if one is to
develop novel and clinically effective inhibitors for cancer
therapy (11, 12, 14-16).
[0780] Melanoma differentiation associated gene-9 (mda-9/syntenin):
a positive regulator of melanoma metastasis: mda-9, also known as
syntenin (1) (mda-9/syntenin), was cloned using subtraction
hybridization as a gene displaying differential expression as a
consequence of induction of irreversible growth arrest, terminal
differentiation and loss of tumorigenic potential in HO-1 human
metastatic melanoma cells following treatment with fibroblast
interferon (IFN) and the protein kinase C activator mezerein (17).
Bioinformatics data employing genome-wide expression datasets (such
as TCGA and GEO) indicate that mda-9/syntenin (syndecan binding
protein; SDCBP) is elevated in numerous cancer types and expression
is enhanced as these cancers progress to a more invasive and
metastatic state. This includes glioblastoma multiforme (GBM) (18),
urothelial carcinomas (19), melanoma (2-4, 20-22), prostate
carcinoma, and hepatocellular carcinoma (HCC). Although both
bioinformatics analysis and our preliminary observations indicate a
positive correlation of mda-9/syntenin expression with multiple
cancers, its exact role(s) in many of these neoplasms have not been
established. Suppression subtractive hybridization between poorly
invasive/nonmetastatic and an invasive/metastatic breast cancer
cell line identified mda-9/syntenin overexpression in metastatic
cells (6). Forced expression of mda-9/syntenin resulted in
increased migration and invasion by normal and non-metastatic
cancer cells, and correlated with a more polarized distribution of
F-actin and increased pseudopodia formation (1, 2, 23).
Additionally, immunohistochemical analysis revealed a statistically
significant gradual increase in MDA-9/syntenin protein expression
from acquired melanocytic nevi to primary melanoma without or with
progression to metastatic melanomas. Thus, the accumulated data
strongly support the hypothesis that mda-9/syntenin functions as a
positive regulator for melanoma metastasis as well as
aggressiveness in melanoma, GBM, HCC, prostate, breast and gastric
cancers, and might be involved in metastasis in multiple additional
cancers.
[0781] The PDZ domains of MDA-9/Syntenin: prevalence and
significance in metastasis: PDZ domains (an acronym for three
proteins-postsynaptic density protein PSD95/SAP90, drosophila tumor
suppressor DLGA, and tight junction protein ZO-1 containing
proteins) are a diverse group of over ISO proteins that control
diverse and central physiologic processes (24-26). One of the
best-studied proteins from this group MDA-9/Syntenin (8-10, 19, 22,
27, 28), has a surprising variety and diversity of interacting
partners and through its specific localization, controls a variety
of molecular events. Structurally, MDA-9/Syntenin has two PDZ
domains: PDZ1 (a.a. 110-193) and PDZ2 (a.a. 194-274) and in the
context of melanoma metastasis, both PDZ domains are critically
involved, since deletion mutants (either one of the two PDZ
domains) of MDA-9/Syntenin significantly reduced lung metastases
compared with the cells transfected with wild type MDA-9/Syntenin
(4). Mechanistically, MDA-9/Syntenin was shown to interact with
c-Src, a member of the Src family tyrosine kinases involved in
numerous biological processes associated with cytoskeletal
organization, including increased cell motility, invasiveness, and
survival (29) through the carboxylate-binding loop of PDZ2.
However, PDZ1 also plays a critical role in binding by promoting
the proper folding of PDZ2 that assembles MDA-9/Syntenin into a
multimeric complex resulting in a more stable functional unit.
Accordingly, MDA-9/Syntenin through its interaction with itself and
with c-Src enables c-Src/FAK signaling complexes clustered at high
concentrations on the plasma membrane to amplify signaling through
FAK intermolecular autophosphorylation. These events lead to
enhanced cell motility, invasion, and metastasis. Accordingly,
loss-of-function (LOF) studies, using genetic and pharmacological
approaches, indicate that inhibiting MDA-9/Syntenin or c-Src and
thereby decreasing the interaction between MDA-9/Syntenin and
c-Src, block metastasis in human melanoma cells (5).
[0782] Clinical Relevance of MDA-9/Syntenin Overexpression:
[0783] Previous studies have shown a statistically significant
increase in MDA-9/syntenin expression in advanced stages of
melanoma, GBM, breast, urothelial, HCC and prostate cancer (6, 7,
9, 19)
[0784] Targeting PDZ domains of MDA-9/Syntenin for pharmacological
intervention. Recent years have witnessed a growing interest in
targeting this class of proteins with some initial success (Table
3) using small peptides, natural products and small molecules.
These initial accomplishments are quite exciting and suggest that
the PDZ domains are "draggable". However, there are several
challenges associated with targeting PDZ domains for therapeutic
purposes (35-42), since they are involved in many protein-protein
interactions. While there are over ISO PDZ domains in the human
genome discovered thus far, their binding surfaces are likely
distinct. Bioinformatics analysis of The Cancer Genome Atlas (TCGA,
cancergenome.nih.gov/) indicates that from at least 1S1
PDZ-containing proteins present in the human genome, many are
overexpressed in multiple cancers, and many of these cancers also
have elevated mda-9/syntenin expression. In turn, each of these
genes has potential interaction partners that can be identified
using protein-protein interaction databases. Peptide-based
inhibitors might suffer from bioavailability limitations, such as
metabolic stability and unfavorable physiochemical properties,
which could reduce their therapeutic benefit. To overcome such
limitations, modifications of peptide inhibitors, such as
.beta.-strand peptide-mimetic and cyclic peptides, for PDZ
protein-protein interactions were suggested (38,43). Nevertheless,
the development of small organic molecules remains the most
promising approach (31). For this reason, we propose to use a
combination of powerful Fragment- and NMR-Based Drug Discovery
(FBDD) approaches (44-47) to derive focused libraries of
MDA-9/Syntenin PDZ1-targeting ligands. Our preliminary data
demonstrates that these strategies enabled the identification and
initial optimization of small molecules capable of targeting and
antagonizing MDA-9/Syntenin in vitro in cell cultures through its
PDZ1 domain (PDZ1in). Interestingly, these small molecules do not
target the PDZ2 domain of MDA-9/Syntenin, demonstrating that
despite the similar global fold, these domains tend to have fairly
distinct binding surfaces.
TABLE-US-00003 TABLE 3 PDZ domains targeted for pharmacological
use. PHASE OF DEVELOP- TARGET ANTAGONIST MENT FUNCTIONS PSD.95
PDZ2/NMDA or nNOS NA-1 Lead Phase 1 Treatment of ischemic brain
damage: Na-1 showed safety and tolerability after IV administration
(30). Dishevelled PDZ/Fz7 Wnt receptor ##STR00066## Lead compound
Induction of apoptosis in human cancer cell lines and tumor growth
inhibition in a mouse xenograft model (31). PSD.95 PDZ2/NMDA or
nNOS Flavonoids Natural product Flavinoids bind to the PSD.95 PDZ2
(32). Dishevelled PDZ/Fz7 Wnt receptor Fz7 peptide (GSKTLQSWRRYH)
Dapper peptide (SGKLKLMTTV) Pre-clinical In Xenopus embryos, Fz7
(or Dapper) peptide attenuates Wnt3A-induced canonical Wnt
signaling (33). Dishevelled PDZ/Fz7 Wnt receptor ##STR00067##
Pre-clinical In Xenopus embryos, NSC-668036 inhibits the canonical
Wnt signaling induced by Wnt3A (34). ##STR00068##
[0785] PDZ1in: small molecules targeting the PDZ1 domain of
MDA-9/Syntenin. A series of compounds have been developed targeting
PDZ1 using a combination of FBDD techniques guided by in silico
docking and NMR-based design. Application of these approaches
generated PDZ1 inhibitory compounds (PDZ1in).
[0786] The bi-dentate compound of this series represented by 113B7
has been characterized using NMR techniques and it displays a
dissociation constant in the low micromolar range according to
NMR-titration binding assays and ITC. interestingly, this compound
spans both the first PDZ domain and the interface between the two
domains (FIG. 52), but it does not appreciably bind to the second
PDZ domain or other PDZs, thereby showing selectivity (see FIG. 52)
and further corroborating our central hypothesis that it is
possible to obtain high affinity, selective PDZ antagonists. The
effect of 113B7 on invasion was tested using a panel of human
cancer cells derived from different sites, including melanoma,
pancreas, prostate, brain (glioblastoma (GBM)), breast, and
hepatocellular carcinoma (HCC). These cancer cell types were chosen
because they display a high basal level of MDA-9/Syntenin
expression at both mRNA and protein levels compared with
corresponding primary normal cell counterparts. Pre-treatment with
113B7 (PDZ1in) significantly inhibited the invasion of all of these
cancer cells. It is worth noting that the compound did not show any
significant toxicity, to melanocytes when tested up to 100
.mu.M.
[0787] The immediate objectives are to comprehend how a novel
metastasis-associated adapter protein, MDA-9/Syntenin, induces
tumor progression culminating in acquisition of metastatic
competence and to develop novel targeted therapeutic agents through
exploitation of elegant approaches using fragment- and
structural-based strategies combined with in silica drug design and
medicinal chemistry. The central hypothesis is that through its two
PDZ domains, MDA-9/Syntenin facilitates tumor progression by
augmenting several distinct but complementary components of the
tumorigenic process, including invasion/migration (2) and
angiogenesis (3), through its ability to activate multiple signal
transduction pathways.
[0788] We propose to derive novel, focused compound libraries
targeting MDA-9/Syntenin using a combination of FBDD techniques
guided by in silica docking and NMR-based design. We will use
initial scaffold fragments and subsequent NMR studies to design and
prioritize compounds to be synthesized and tested iteratively. The
main objective is identify compounds that specifically bind to the
PDZ1 domain.
[0789] In all binding assays, the MDA-9/Syntenin construct used for
the screen contains both PDZ domains. Constructs uniformly labeled
with .sup.15N and .sup.13C or .sup.13C-Met selectively labeled are
used for binding, mapping and structural studies. These same
proteins are used for ITC binding studies.
[0790] As anticipated, our fragment-screen of 5,000 compounds
resulted in only a few binding hits (tested at 500 .mu.M against
PDZ1/2 at 10 .mu.M using ID .sup.1H NMR aliphatic screen followed
by 2D [.sup.15N, .sup.1H] HSQCs titration and ITC measurements).
Interestingly, we found that these hits bind only to the PDZ1
domain or to the space between the domains, while no binders for
the PDZ2 were identified.
[0791] Based on these studies and NMR guided initial docking
calculations, >50 bi-dentate compounds were synthesized and
tested, leading to compound 113B7 (FIG. 52). These initial
higher-affinity bi-dentate compounds represented by 113B7 (FIG. 52)
do not significantly bind to the second domain up to 100 .mu.M,
suggesting that it is indeed possible to obtain high affinity and
selective ligands with the proposed approach, (4). Moreover, as a
way to further address specificity of our ligands, we also tested
the most promising compounds against two closely related domains to
the PDZs of MD A-9/Syntenin, such as the PDZ tandem protein
Harmonin (36% identity to PDZ2 of MDA-9/Syntenin and 31% to PDZ1 of
MDA-9/syntenin) or PDZ domain from X11/mint scaffold protein (33%
identity with PDZ1), and no significant binding to these
counter-screen proteins was observed (FIG. 52).
[0792] In addition, the molecules are tested against a series of
proteins available in the laboratory involved in protein-protein
interactions including Mc1-1, Bc1-xL, Bid, MT1-MMP-PEX domain,
IL20R, RKIP, Jnk2 (JIP-binding site Met labeled), Ubc13, SIAH1, and
Ship2-SAM. Preliminary studies with several of these protein
domains and 113B7 revealed no appreciable binding at 50 .mu.M with
1:1 ligand/protein ratio. Based on these data, we propose to derive
a small library of 113B7 analogues, guided by NMR structural data
and test these resulting compounds iteratively against
MDA-9/Syntenin by NMR and ITC. In designing compounds using
elements of the libraries, particular emphasis will be put on
drug-like properties of the compounds. In order to prioritize the
compounds for further cellular and efficacy studies, in vitro
ADME-tox studies will be performed to select those candidates with
the highest likelihood to possess necessary stability, solubility
and cell permeability to be meaningfully tested in cells and in
vivo. Additionally, the MTD and PK properties on selected compounds
prior to their eventual evaluations in vivo in will be addressed.
Preliminarily, PK studies with 113B7 indicate that the compound is
stable and long-lived with nearly 100% bioavailability when
administered IP (FIG. 53).
[0793] Synthetic Chemistry and Design:
[0794] In designing the compounds to be tested and/or synthesized,
particular attention will be given to the chemical properties of
the selected compounds in an attempt to address "drug-likeness" on
empirical grounds. The goal in using these empirical drug-like
property filters is to predict favorable outcome in ADME-Tox
(adsorption, distribution, metabolism, excretion, toxicity)
studies, as well as final success as drugs in humans. In performing
small-scale synthesis of the initially proposed linked compounds,
we will make extensive use of either solid-phase synthesis or
solution-phase synthesis aided by resin-bound reagents and
scavengers. For example, basic reducing reactions including
reductions of carbonyl compounds, azides and oximes, reductive
animation, reduction of conjugated enones to unsaturated alcohols,
will be performed with MP-Borohydride resin (Argonaut). Similarly,
our typical synthesis of acyl or sulfonyl derivatives, including
esters, amides, and sulfonamides will be performed by using PS-DMAP
for "Catch and Release" reactions, (Argonaut).
[0795] Likewise, we have successfully performed several reactions
in homogenous media including Knoevenagel condensations,
Diels-Alder, Claisen rearrangements, etc. For compounds'
purification, we will make extensive use of precision packed
RediSep columns and our CombiFlash Companion flash-chromatography
systems. All final compounds are generally purified to >95%
purity, as determined by a HPLC Breeze from Waters Co. using an
Atlantis T3 3 .mu.M 4.6 mm.times.150 mm reverse phase column.
Compounds for in vivo studies are purified to .gtoreq.95% purity
using preparative HPLC. Based on our initial SAR studies on
compound 113B7 (Table 4) we propose a number of possible
derivatizations.
TABLE-US-00004 TABLE 4 Examples of SAR studies (out of >50
compounds synthesized and tested thus far). Dissociation constants
are estimated by NMR titration in 2D HSCQ experiments. ##STR00069##
K.sub.d, % Comp R1 .mu.M.sup.a migr..sup.b 113B7 ##STR00070## ~10
62 112H9 ##STR00071## 38 24 112H10 ##STR00072## ~40 35
[0796] Structural Studies on MDA-9/Syntenin Inhibitor
Complexes:
[0797] While molecular docking studies in conjunction with chemical
shift mapping will provide valuable insights on the mode of binding
of compounds, we plan to obtain a high-resolution NMR structure of
the most promising compound in complex with MDA9/syntenin, which
may provide critical information for proper positioning of a
chemical linker or further optimizations and scaffold hopping. This
potentially time consuming task is largely facilitated by our
current knowledge of the NMR solution structure of the PDZ1 domain
of MDA-9/Syntenin and the availability of the complete resonance
assignments. In order to obtain high-resolution structural
information on MDA-9/Syntenin-inhibitor complexes, the combination
of 3D .sup.15N, .sup.13C-edited NOESY [.sup.1H-.sup.1H] and 3D
.sup.15N, .sup.13C-filtered [.sup.1H-.sup.1H] NOESY experiment
supported by two-dimensional (2D) [.sup.1H-.sup.1H] NOESY
experiments in D20, should provide necessary intra- and
inter-molecular NOE constraints for structure calculation.
Structure calculation will be obtained using a modified CYAN A (80)
and energy minimizations and structure-based docking and analyses
will be performed using Sybyl-X (Cetera, N.C.) and GOLD (CCDC).
[0798] SARs of Small Molecule Lead Inhibitors of MDA-9/Syntenin
Using Cellular Assays:
[0799] A critical step in assessing the properties of the compounds
and to support iterative optimization will consist of a number of
cellular assays that are relevant to MDA-9/Syntenin activity. For
example, 113B7 targeting only PDZ1 domains shows very strong
anti-invasive properties against human melanoma and genetically
modified (mda-9/syntenin overexpressing) immortal human melanocytes
(FM-516-mda-9) according to the manufacturer's instruction.
[0800] Three different concentrations will be used for each
compound and the small molecule that displays reproducible,
dose-dependent reduction of invasion in both cell lines will be
further validated using different in vitro approaches including
Boyden Chamber assays, anchorage-independent growth in soft agar
and migration assays. The molecular mechanism(s) of anti-invasive
properties, in particular the effects on MDA-9/Syntenin downstream
signaling pathways such as c-Src phosphorylation and FAK. P38 MAPK
and NF-.kappa.B activation will be scrutinized using our previously
described approaches (2-5, 110, 111).
[0801] Effect of PDZ1In on c-Src Binding to MDA-9/Syntenin:
[0802] Another fundamental issue that we will address as part of
the proposed hit-to-lead optimization process is the specificity of
the binding of 113B7 to the PDZ1 domain of MDA-9/Syntenin. Previous
studies demonstrate that c-SRC initially binds to MDA-9/Syntenin
through the PDZ2 domain followed by recruitment of the PDZ1 domain
(4. 5). Deletion analysis indicated that both PDZ1 and PDZ2 domains
of MDA-9/Syntenin are necessary for retention of binding and
downstream signaling and deletion of either or both of the PDZs
results in a loss of MDA-9/c-SRC interaction and inhibition of
transformation-associated properties (invasion, growth in agar) in
vitro and metastasis of melanoma cells to the lungs of mice in vivo
(4, 5). To determine if the PDZin molecule alters physical binding
we performed co-immunoprecipitation analysis with c-Src and
MDA-9/Syntenin in the presence or absence of 113B7 (FIG. 54). In
the case of 113B7, which binds to PDZ1 and the interdomain of
MDA-9/Syntenin, no inhibition of c-Src binding was evident. This
results suggests that other pathways are regulated by
MDA-9/Syntenin that contribute to metastasis when PDZ1is inhibited
by 113B7 (FIG. 54).
[0803] In Vitro ADME-Tox Properties and Pharmacokinetks
Studies:
[0804] Studies will be conducted for measurements of plasma and
microsomal stability, and protein binding. Using two API Sciex-4000
quadrupole mass spectrometers, each with a linear ion trap and
coupled to a Waters Aquity ultra-performance liquid chromatograph.
Initial rapid assessment of compound exposure in a series of
candidates will be conducted by using the RACE protocols adapted by
the facility (see also Vertebrate Animals). Subsequently, most
promising compounds will be subjected to a full
bioavailability/distribution analysis. Tissue penetration of
compounds will be determined in a non-quantitative manner by LC/MS.
These data will give us a rough idea of the concentration of intact
compound that reaches the blood stream and the lungs, allowing us
to compare that result with the concentration proven to be
effective in culture experiments and the preliminary in vivo
studies where the drugs are administered IV, IP or orally, and thus
informing us whether we are achieving sufficient levels of compound
in the desired target tissue. For example, preliminary PK studies
with 113B7 were conducted (FIG. 53) suggesting that IP
administration (a route of administration for repeated doses for
initial efficacy studies) is a viable route of administration for
PDZ1in.
[0805] To demonstrate in vivo activity and efficacy, compounds must
meet certain minimum ADME criteria. As part of this grant
application, we will select and iteratively refine the structure of
compounds with favorable ADME profiles. Ultimately, these
activities will guide the choice of possible antagonists as
pharmacological tools for further in vitro cell and in vivo
pharmacology and efficacy studies thus minimizing side reactions
and liabilities that may be intrinsic with the derived molecules
and not related with inhibition of the target. Possible problems
that will be identified in any particular series of compounds will
be addressed and possibly eliminated iteratively in designing
additional compounds. The iterative process of optimizations and
evaluation will include our well established and straightforward
go-no-go decision tree in which potency, selectivity and drug
likeness (e.g., Kit<500 nM: high solubility in buffer
S>10.times.K.sub.d) as determined by an NMR method; selectivity
against the counter screen PDZs and other PPIs listed above when
tested at 10.times.K.sub.d; measured favorable ADME-T properties
with T1,2 in plasma and microsomes >30 minutes; cell
permeability) are iteratively confronted with cellular mechanism
based activity, favorable pharmacological properties and
ultimately, in vivo efficacy. These studies will include measuring
cytotoxicity against normal cells (melanocytes, hepatocytes,
prostate epithelial cells. HUVEC cells), determining inhibition of
invasion and cellular interaction with defined partners, PK studies
followed by in vivo efficacy with animal models.
[0806] Mechanistically Evaluate Current MDA-9/Syntenin PDZ1in in
Preventing Melanoma Cell Adhesion, Invasion and Metastasis and
Identify/Characterize the Next Generation of PDZ1in.
[0807] As emphasized, mda-9/syntenin is a pro-metastatic gene and
genetic or pharmacological inhibition of this gene, its interacting
partners or its downstream regulated pathways suppress the cancer
phenotype in vitro and in vivo in animal models. Additionally,
through regulation of several pro-angiogenic factors.
MDA-9/syntenin promotes angiogenesis thereby facilitating
metastasis (111). These pre-clinical observations strongly support
a fundamental role of MDA-9/Syntenin in various stages of tumor
cell spreading (migration and invasion) and metastasis, which is a
major cause of cancer-associated death. Metastasis is a complex
process (112) involving intravasation of tumor cells from the
primary tumor site (involving degradation and passage through the
basement membrane), survival in the systemic circulation, adherence
at a secondary target site, extravasation at the secondary site and
creation of a favorable environment for expansion (which also
involves angiogenesis (113)). In this multistep process, adherence
is essential and directly correlates with the metastatic capacity
of tumor cells. In our studies (FIG. 44), we demonstrate that
MDA-9/Syntenin regulates the adhesion and invasion phenotypes of
tumor cells, which are blocked by 113B7 (PDZ1ln).
[0808] PDZ1In Inhibits B16 Experimental Metastasis.
[0809] Preliminary studies were performed to determine if
administering PDZ1ln (113B7) would affect experimental metastasis.
B16, a highly aggressive metastatic mouse melanoma, was
intravenously injected resulting in lung nodules within 3 weeks. In
this model, repeated treatment LP. with small molecule PDZ1in
significantly reduced the number of lung nodules (FIG. 57) in
comparison with the control group, which received vehicle. This
experiment confirms that treatment with PDZ1in targeting
MDA-9/Syntenin can directly prevent formation of lung metastases in
vivo.
[0810] Expression of MDA-9/Syntenin Modulates the Adhesion Ability
of Metastatic Tumor Cells in the Lungs:
[0811] To establish that MDA-9/Syntenin modulates cancer cell
homing and adhesion to secondary sites such as the lungs, an early
event that correlates with lung metastatic potential, we suppressed
MDA-9/Syntenin expression (using an adenovirus expressing an
shmda-9 construct (111)) in aggressive metastatic human MeWo
melanoma cells (expressing luciferase; MeWo-Luc) and directly
injected cells I.V. into mice and visualized tumor cell
homing/adhesion to the lungs by bioluminescence (BLI) imaging (FIG.
58). Mice inoculated with Ad. shmda-9 pre-infected cells were
present at lower levels than control groups in the lungs
immediately after injection (within 45 min post-inoculation),
suggesting that MDA-9/Syntenin expression might be critical for
adhesion ability of potentially metastatic melanoma (and
potentially other circulating tumor) cells, which in principle,
could ultimately have a direct effect on development of metastatic
lesions at secondary sites. To evaluate the anti-metastatic
efficiency of 113B7 (PDZ1in) we inoculated metastatic MeWo-Luc
cells I.V. and treated animals with either DMSO (compound solvent)
or test compound for four weeks by IP injection. After 40 days lung
metastases were imaged BLI (FIG. 59).
[0812] MDA-9/Syntenin Modulates Adhesion-Related Gene Expression in
Human Melanoma:
[0813] Our initial experiments confirmed that interruption of
MDA-9/Syntenin-mediated signaling pathways significantly reduced
the ability of melanoma cells to adhere in the lungs of animals
(FIG. 58). To begin to understand the molecular basis of this
phenomenon we conducted a PCR-based array containing
adhesion-related genes (SA Bioscience) (FIG. 56). RNA profiling
between parental aggressive C8161.9 human melanoma cells and its
shmda-9 expressing clones identified changes in the levels of
several important adhesion-associated genes including Integrin
.beta.2, MMP-2. MMP-12 and MMP-14 (FIG. 56). The relevance of these
changes to melanoma metastasis as mediated by MDA-9/Syntenin and
the possible effect of our proof-of-concept small molecule PDZ1in
(113B7) will be explored.
[0814] Validation of Genes Involved in MDA-9/Syntenin-Mediated
Adhesion:
[0815] As shown in FIG. 56, an adhesion array was screened using
C8161.9 human melanoma cells and its shmda-9 expressing clone (111)
documenting changes in expression of specific signature genes
associated with adhesion. This will first be validated be validated
in melanoma cell lines and melanocytes expressing MDA-9/Syntenin to
establish a direct correlation between MDA-9/syntenin expression
and adhesion molecule expression. To define potential clinical
relevance, we will perform immunohistochemistry for these genes in
patient-derived melanoma samples, which are commercially available
through Imgenex Corp. (San Diego, Calif.) Correlations between the
expression levels of MDA-9/Syntenin and the analyzed candidate
proteins will be established by a two-sided Mann-Whitney test
[0816] Confirming the Involvement of Specific Candidate Genes in
MDA-9/Syntenin-Augmented Adhesion:
[0817] Through gain-of-function (GOF) and loss-of function (LOF)
analysis as described (111), we will develop a series of cell lines
expressing different candidate genes or shRNAs to these genes in
luciferase expressing primary immortal melanocytes and aggressive
melanoma cells, respectively. The adhesion ability of cells in
which target genes have been manipulated will be investigated using
our in vivo lung retention model (FIG. 57).
[0818] Identify the signal transduction pathways downstream of
MDA-9/Syntenin regulating adhesion-related genes: Once validated,
the next step will be to define the signaling cascade that
regulates the expression of appropriately altered adhesion gene(s).
The generation of these molecules may involve activation of a
signaling cascade(s) we have already identified (i.e. NF-.kappa.B,
Akt, HIF-1.alpha.) or it might involve additional signaling
pathways, such as Ephrin/IGF1/STAT3 and their receptors. Using
signal pathway-specific arrays as previously described (2-5). we
will define appropriate signaling pathways modified as a
consequence of MDA-9/Syntenin regulation and then mechanistically
define their role in regulating adhesion of melanoma cells.
[0819] Evaluate the Therapeutic Efficacy of PDZ1in to Inhibit Lung
Metastasis Development in a Transgenic Melanoma Metastasis Mouse
Model:
[0820] A conditional mouse model, Braf.sup.V600E-induced/Pten
deficient (114), which recapitulates the key pathophysiological
aspects of human melanoma and shows short latency with metastases
in lymph nodes and lungs will be evaluated with our lead small
molecule. The utility of this model in pre-clinical experimental
therapeutics has been demonstrated (114). We have also confirmed
that tumors developing in this animal express MDA-9/Syntenin (FIG.
60). Founder animals for generating this model were acquired from
Jackson Laboratories and colonies have been expanded. Studies will
confirm that specific PDZ1in have therapeutic activity in this
mouse model. For these experiments, a group of S mice will be
treated with our test compounds before melanoma initiation in
adults by topical administration with 4-hydroxytamoxifen (4-HT). In
the absence of 4-HT, mice display no discernible phenotype.
However, topical or systematic administration with 4-HT results in
the rapid development of melanoma with lung metastasis and animals
require euthanasia within 3-6 weeks. In other experiments, melanoma
will be initiated first and after one week, when the invasive
properties are generally observed, they will be treated (IP, 6
injections, 3.times. a week, for 2-week) with our test compounds.
In both cases, Kaplan Meier survival curves will be used to compare
the efficacy of our test materials. In addition, we will also
collect the lungs for pathological evaluation.
[0821] Go-No-go Decision for the Next Generation MDA-9/Syntenin
PDZ1in:
[0822] We have defined a specific path for moving forward for in
vivo testing of newer PDZ1in in melanoma in and then for in vivo
testing. Initial hits will first be screened for chemical binding
properties to the PDZ1 domain of MDA-9/syntenin. Potentially
promising molecules (e.g., with K.sub.d<500 nM), will be
stratified based on drug-like properties, e.g. i) high solubility
in buffer (S>10.times. Kd as determined by NMR); ii) favorable
ADME-T properties (T.sub.1/2 in plasma and microsomes >30
minutes); iii) selectivity against counter-PDZs and additional
proteins from the protein-protein interactions panel when tested at
10.times.K.sub.4 The chemicals will be screened for toxicity using
various normal cells including primary melanocyte NHEM (From ATCC),
immortal melanocytes FM-516 cells, RWPE-1 prostate epithelial cells
(ATCC), hepatocytes, IM-PHFA immortal astrocytes and human
endothelial cells (ATCC)). MTT and trypan blue dye exclusion assays
will be performed using a concentration >10.times. of the
K.sub.d. Any compound displaying toxicity below this range will be
excluded from further evaluation. Next, we will confirm the
specificity of candidate compounds. MDA-9/Syntenin overexpressing
tumor cells or engineered normal cells will be treated with
compound followed immunoprecipitation and confocal microscopy to
monitor interactions between MDA-9/Syntenin with specific proteins
(e.g., c-Src, AEG-1 and IGF-1R for melanoma, liver and prostate
cancer cells, respectively). In vitro invasion assays will also be
done to determine the anti-invasive properties of candidate PDZ1in
molecules. First, we will inject test PDZ1in-pre-treated MeWo-Luc
cells I.V. and follow lung retention over time by BLI (115-117). We
will also compare the metastatic potency of non-treated cells vs.
pre-treated cells. These experiments will be very informative for
defining and establishing the molecular mechanism of drug action in
the context of adhesion, an early and compulsory step in
metastasis, invasion and colonization, later events in metastasis.
In addition, we are also proposing to investigate the efficacy of
our small molecules in two different contexts, a) protection of
animals from developing metastasis resulting from circulating
metastatic melanoma cells; and b) clinical benefits against
established metastases. To define the protective role of lead
compounds in preventing metastases (a) mice will be pre-treated
with drugs before IV injection of MeWo-luc cells. After injection
of cancer cells, mice will be randomly separated into two
experimental groups and one group will continue without any post
treatment while the other set will receive experimental drugs via
intraperitoneal injection (I.P.) for a total of 9 injections (dose
will be determined on the basis of MTD and pharmacokinetics data).
BLI imaging will be performed periodically to track metastasis
development and to determine therapeutic efficacy. Kaplan Meier
survival curves will be generated to determine the efficacy of the
therapeutic in protecting animals from developing metastases. In
the second experimental protocol (b), we will first inject the
cells and allow time for metastases to develop, which will be
detected by BLI, before initiating treatment. Once lung metastases
are detected by BLI imaging, animals will be treated with test
compound or untreated for control animals. Anti-metastatic
potential of lead compounds against established metastases will be
monitored by BLI, and quantified using Kaplan Meier survival
curves. In addition to immunocompromised mice, we would like to
address similar questions regarding the efficiency of our molecules
in syngeneic animal models or spontaneous metastatic model in
melanoma. HCC and prostate cancer. Therapeutic efficacy of the
PDZ1in will also be evaluated using imaging approaches, BLI and
SPECT, for these three cancer indications.
[0823] The observation that MDA-9/Syntenin augments multiple known
genes involved in adhesion implies that one or multiple genes are
essential in mda-9/syntenin-mediated melanoma cell adhesion. Using
GOF and LOF approaches we will confirm the role of candidate genes
in mediating in vivo adherence as defined by reduced lung retention
and metastatic nodule formation in vivo. In addition, by employing
different chemical and genetic inhibitors of various signaling
molecules we will be able to untangle the molecular mechanism(s)
underlying these gene regulation changes. As shown in our data, we
confirm that MDA-9/Syntenin is directly involved in invasion and
metastasis and represents a viable target for inhibiting this
process. We predicted and subsequently confirmed that
MDA-9/Syntenin PDZ1-targeted small molecule inhibitors (e.g.,
PDZ1in) can reduce melanoma cell invasion in vitro, alter retention
of metastatic cells in the lungs and inhibit the development of
lung metastases.
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TABLE-US-00005 [1164] INFORMAL SEQUENCE LISTING (PDZI domain) SEQ
ID NO: 1 QGIREVILCKDQDGKIGLRLKSIDNGIFVQLVQANSPASLVGLRFGDQVL
QINGENCAGWSSDKAHKVLKQAFGEKITMTIRDR
Provisional Embodiments
[1165] Embodiments contemplated herein include the following.
[1166] The following definitions pertain exclusively to Provisional
(i.e. P) embodiments.
[1167] The term "alkyl" refers to linear (unbranched) or branched
chain unsubstituted hydrocarbon groups of about 1 to 20 carbon
atoms, for example. The term "substituted alkyl" refers to an alkyl
group substituted by, for example, about one, two three or four
substituents, examples of which include but are not limited to:
halo, hydroxy, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino,
alkylamino, substituted alkylamino, cycloalkylamino, substituted
cycloalkylamino, arylamino, substituted arylamino, aralkylamino,
substituted aralkyamino, disubstituted amines in which the 2 amino
substituents are selected from alkyl, aryl or aralkyl;
alkanoylamino, aroylamino, aralkanoylamino, substituted
alkanoylamino, substituted arylamino, substituted aralkanoylamino,
thiol, alkylthio, arylthio, aralkylthio, alkylthiono, arylthiono,
aralkylthiono, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl,
sulfonamido, e.g. SO.sub.2NH.sub.2, substituted sulfonamido, nitro,
cyano, carboxy, carbamyl, e.g. CONH.sub.2, substituted carbamyl
e.g. CONHalkyl, CONHaryl, CONHaralkyl or cases where there are two
substituents on the nitrogen selected from alkyl, aryl or aralkyl;
alkoxycarbonyl, aryl, substituted aryl, guanidino, heterocyclyl,
e.g., indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl,
pyridyl, pyrimidyl, pyrrolidinyl, piperidinyl, morpholinyl,
piperazinyl, homopiperazinyl and the like, and substituted
heterocyclyl. These substituents may be further substituted, e.g.
with alkyl, alkoxy, aryl, aralkyl, etc.
[1168] The term "aryl" refers to compounds which contain an
aromatic group, e.g. a monocyclic or polycyclic aromatic compound.
Monocyclic aryls generally have about 4 to about 7 carbon atoms,
bicyclic aryls may have e.g. from about 7 to about 11 carbon atoms,
and tricyclic aryls may contain from about 10 to about 15 or more
carbon atoms. Exemplary aryls are or comprise groups that include
but are not limited to: phenyl, naphthyl, biphenyl (diphenyl),
thienyl, indolyl, etc. Aryls may be substituted or unsubstituted,
and may or may not include one or more heteroatoms (e.g. S, N, O,
etc.) in one or more ring structures (heteroaryls).
[1169] The term "arylalkyl" refers to an aryl or a substituted aryl
group bonded directly to an alkyl group, such as benzyl.
[1170] The term "substituted aryl" refers to an aryl group
substituted by, for example, about one to about four (e.g. 1,2, 3,
or 4) substituents such as alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, aralkyl, halo, trifluoromethoxy, trifluoromethyl,
hydroxy, alkoxy, alkanoyl, alkanoyloxy, aryloxy, aralkyloxy, amino,
alkylamino, arylamino, aralkylamino, dialkylamino, alkanoylamino,
thiol, alkylthio, ureido, nitro, cyano, carboxy, carboxyalkyl,
carbamyl, alkoxycarbonyl, alkylthiono, arylthiono,
arylsulfonylamine, sulfonic acid, alkysulfonyl, sulfonamido,
aryloxy and the like. The substituent may be further substituted by
hydroxy, halo, alkyl, alkoxy, alkenyl, alkynyl, aryl or
aralkyl.
[1171] Embodiment P1. A compound of Formula 1
##STR00073##
wherein Y and X are independently selected from the group
consisting of: --CONH--, --NHCO--, --SO.sub.2NH--, --O--, --CH2-,
--S--, --SO.sub.2--, --NH--, --N(CH.sub.3)--, --COSO.sub.2NH--,
--OCH.sub.2--, and --CH.sub.2O--; R1 is --H or independently mono,
di-, tri- or tetra-substituted with any of the following: --F,
--Cl, --Br, --CF.sub.3, --OCF.sub.3, --OCH.sub.3, --CH3,
--CH.sub.2CH.sub.3, --CONH.sub.2, --NHCOH, --SO.sub.2NH.sub.2,
--OH, --NH.sub.2, --NH(CH.sub.3), --N(CH3)2 --COSO.sub.2NH.sub.2,
--CH.sub.2OH, --CH.sub.7CH1OH; and R2 is a substituted or
un-substituted aryl or a substituted or un-substituted
alkyl-aryl.
[1172] Embodiment P2. The compound of embodiment P1, wherein said
compound is Formula II
##STR00074##
[1173] Embodiment P3. A salt or prodrug of any of the compounds of
embodiments P1-P2.
[1174] Embodiment P4. A method of preventing or treating cancer in
a subject in need thereof, comprising administering to the subject
a therapeutically effective amount of any of the compounds of
embodiments P1-P3, wherein the therapeutically effective amount is
sufficient to prevent or treat the cancer.
[1175] Embodiment P5. A method of sensitizing cancer cells to
killing by radiation, comprising contacting the cancer cells with a
therapeutically effective amount of any of the compounds of
embodiments P1-P3, wherein the therapeutically effective amount is
sufficient to sensitize the cancer cells to killing by
radiation.
[1176] Embodiment P6. A method of slowing or preventing metastasis
of cancer cells in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
any of the compounds of embodiments P1-P3, wherein the
therapeutically effective amount is sufficient to slow or prevent
the metastasis.
[1177] Embodiment P7. The method of embodiment P6, wherein the
therapeutically effective amount is sufficient to slow or prevent
at least one of invasion, migration, and angiogenesis.
[1178] Embodiment P8. The method of any of embodiments P4-P6,
wherein the cancer is selected from the group consisting of
glioblastoma multiforme, melanoma, prostate, breast, and liver
cancer.
[1179] Embodiment P9. A method of treating a glioblastoma
multiforme brain tumor in a subject in need thereof, comprising
performing surgery on the subject to debulk the glioblastoma multi
forme brain tumor; radiosensitizing remaining tumor cells by
administering to the subject a therapeutically effective amount of
at least one of the compounds of any of embodiments P1-P3, wherein
the therapeutically effective amount is sufficient to sensitize the
remaining tumor cells to killing by radiation; and providing
radiation therapy to the subject.
[1180] Embodiment P10. A method of slowing or preventing prostate
cancer metastasis to secondary sites in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of any of the compounds of embodiments P1-P3, wherein the
therapeutically effective amount is sufficient to slow or prevent
the metastasis.
[1181] Embodiment P11. The method of embodiment P10, wherein the
therapeutically effective amount is sufficient to slow or prevent
at least one of invasion, migration, and angiogenesis.
EMBODIMENTS
[1182] Embodiments further contemplated herein include the
following:
[1183] Embodiment 1. A compound, or pharmaceutically acceptable
salt thereof, having the formula:
##STR00075##
wherein R.sup.1 is independently halogen, --CX.sup.1.sub.3,
--CHX.sup.1.sub.2, --CH.sub.2X.sup.1, --OCX.sup.1.sub.3,
--OCH.sub.2X.sup.1, --OCHX.sup.1.sub.2, --CN, --SO.sub.n1R.sup.1D,
--SO.sub.v1NR.sup.1AR.sup.1B, --NHC(O)NR.sup.1AR.sup.1B,
--N(O).sub.m1, --NR.sup.1AR.sup.1B, --C(O)R.sup.1C,
--C(O)--OR.sup.1C, --C(O)NR.sup.1AR.sup.1B, --OR.sup.1D,
--NR.sup.1ASO.sub.2R.sup.1D, --NR.sup.1AC(O)R.sup.1C,
--NR.sup.1AC(O)OR.sup.1C, --NR.sup.1AOR.sup.1C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.1
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.2 is independently hydrogen,
halogen, --CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2, --CN,
--SO.sub.n2R.sup.2D, --SO.sub.v2NR.sup.2AR.sup.2B,
--NHC(O)NR.sup.2AR.sup.2B, --N(O).sub.m2, --NR.sup.2AR.sup.2B,
--C(O)R.sup.2C, --C(O)--OR.sup.2C, --C(O)NR.sup.2AR.sup.2B,
--OR.sup.2D, --NR.sup.2ASO.sub.2R.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O)OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; L.sup.1 is a bond,
--S(O).sub.2--, --N(R.sup.3)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--, --N(R.sup.3)C(O)NH--,
--NHC(O)N(R.sup.3)--, --S(O).sub.2N(R.sup.3)--,
--N(R.sup.3)S(O).sub.2--, --(O)S(O).sub.2N(R.sup.3)--,
--N(R.sup.3)S(O).sub.2C(O)--, --C(O)O--, --OC(O)--, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, or substituted or unsubstituted
heteroarylene; R.sup.3 is independently hydrogen, --CX.sup.3.sub.3,
--CHX.sup.3.sub.2, --CH.sub.2X.sup.3, --CN, --C(O)R.sup.3C,
--C(O)OR.sup.3C, --C(O)NR.sup.3AR.sup.3B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; L.sup.2 is a bond,
--S(O).sub.2--, --N(R.sup.4)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--, --N(R.sup.4)C(O)NH--,
--NHC(O)N(R.sup.4)--, --S(O).sub.2N(R.sup.4)--,
--N(R.sup.4)S(O).sub.2--, --(O)S(O).sub.2N(R.sup.4)--,
--N(R.sup.4)S(O).sub.2C(O)--, --C(O)O--, --OC(O)--, substituted or
unsubstituted alkylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted heterocycloalkylene, substituted or
unsubstituted arylene, or substituted or unsubstituted
heteroarylene; R.sup.4 is independently hydrogen, --CX.sup.4.sub.3,
--CHX.sup.4.sub.2, --CH.sub.2X.sup.4, --CN, --C(O)R.sup.4C,
--C(O)OR.sup.4C, --C(O)NR.sup.4AR.sup.4B, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.1A, R.sup.1B,
R.sup.1C, R.sup.1D, R.sup.2A, R.sup.2B, R.sup.2C, R.sup.2D,
R.sup.3A, R.sup.3B, R.sup.3C, R.sup.4A, R.sup.4B, and R.sup.4C are
independently hydrogen, --CX.sub.3, --CN, --COOH, --CONH.sub.2,
--CHX.sub.2, --CH.sub.2X, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.1A and R.sup.1B substituents
bonded to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; R.sup.2A and R.sup.2B substituents bonded
to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; R.sup.3A and R.sup.3B substituents bonded
to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; R.sup.4A and R.sup.4B substituents bonded
to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl; X, X.sup.1, X.sup.2, X.sup.3, and X.sup.4
are independently --F, --Cl, --Br, or --I; n1 and n2 are
independently an integer from 0 to 4; m1, m2, v1, and v2 are
independently 1 or 2; and z1 is an integer from 0 to 4.
[1184] Embodiment 2. The compound of embodiment 1, wherein R.sup.1
is independently halogen, --CX.sup.1.sub.3, --CHX.sup.1.sub.2,
--CH.sub.2X.sup.1, --OCX.sup.1.sub.3, --OR.sup.2D, --CN,
--NR.sup.1AR.sup.1B, substituted or unsubstituted alkyl or two
adjacent R.sup.1 substituents may optionally be joined to form a
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[1185] Embodiment 3. The compound of embodiment 1, wherein R.sup.1
is independently halogen, --CX.sup.1.sub.3, --CHX.sup.1.sub.2,
--CH.sub.2X.sup.1, --OCX.sup.1.sub.3, --OR.sup.2D, --CN,
--NR.sup.1AR.sup.1B, or substituted or unsubstituted alkyl.
[1186] Embodiment 4. The compound of embodiment 1, wherein R.sup.1
is independently halogen, --OR.sup.2D, or --CH.sub.3.
[1187] Embodiment 5. The compound of embodiment 1, wherein R.sup.1
is independently halogen or --CH.sub.3.
[1188] Embodiment 6. The compound of embodiment 1, wherein R.sup.1
is --CH.sub.3.
[1189] Embodiment 7. The compound of any one of embodiments 1-6,
wherein L.sup.1 is a bond, --S(O).sub.2--, --N(R.sup.3)--, --O--,
--S--, --C(O)--, --C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--,
--N(R.sup.3)C(O)NH--, substituted or unsubstituted alkylene, or
substituted or unsubstituted heteroalkylene.
[1190] Embodiment 8. The compound of any one of embodiments 1-6,
wherein L.sup.1 is a bond, --C(O)N(R.sup.3)--, --N(R.sup.3)C(O)--,
substituted or unsubstituted alkylene, or substituted or
unsubstituted heteroalkylene.
[1191] Embodiment 9. The compound of any one of embodiments 1-6,
wherein L.sup.1 is --C(O)N(R.sup.3)--, unsubstituted alkylene, or
unsubstituted heteroalkylene.
[1192] Embodiment 10. The compound of any one of embodiments 1-6,
wherein L.sup.1 is --C(O)N(R.sup.3)--.
[1193] Embodiment 11. The compound of any one of embodiments 1-6,
wherein L.sup.1 is --C(O)NH--.
[1194] Embodiment 12. The compound of any one of embodiments 1-11,
wherein L.sup.2is a bond, --S(O).sub.2--, --N(R.sup.4)--, --O--,
--S--, --C(O)--, --C(O)N(R.sup.4)--, --N(R.sup.4)C(O)--,
--N(R.sup.4)C(O)NH--, substituted or unsubstituted alkylene, or
substituted or unsubstituted heteroalkylene.
[1195] Embodiment 13. The compound of any one of embodiments 1-11,
wherein L.sup.2is a bond, --N(R.sup.4)C(O)--, or substituted
heteroalkylene.
[1196] Embodiment 14. The compound of any one of embodiments 1-11,
wherein L.sup.2is a bond or substituted heteroalkylene.
[1197] Embodiment 15. The compound of any one of embodiments 1-11,
wherein L.sup.2 is substituted heteroalkylene.
[1198] Embodiment 16. The compound of any one of embodiments 1-11,
wherein L.sup.2is substituted 2 to 6 membered heteroalkylene.
[1199] Embodiment 17. The compound of any one of embodiments 1-11,
wherein L.sup.2is --NHC(O)CH.sub.2CH.sub.2--.
[1200] Embodiment 18. The compound of any one of embodiments 1-17,
wherein R.sup.2 is independently hydrogen, halogen,
--CX.sup.2.sub.3, --CHX.sup.2.sub.2, --CH.sub.2X.sup.2,
--OCX.sup.2.sub.3, --OCH.sub.2X.sup.2, --OCHX.sup.2.sub.2,
--NR.sup.2AR.sup.2B, --C(O)R.sup.2C, --C(O)--OR.sup.2C,
--C(O)NR.sup.2AR.sup.2B, --OR.sup.2D, --NR.sup.2AC(O) R.sup.2C,
--NR.sup.2AC(O)OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[1201] Embodiment 19. The compound of any one of embodiments 1-17,
wherein R.sup.2 is independently hydrogen, halogen,
--NR.sup.2AR.sup.2B, --C(O)R.sup.2C, --C(O)--OR.sup.2C,
--C(O)NR.sup.2AR.sup.2B, --OR.sup.2D, --NR.sup.2AC(O)R.sup.2C,
--NR.sup.2AC(O)OR.sup.2C, --NR.sup.2AOR.sup.2C, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[1202] Embodiment 20. The compound of any one of embodiments 1-17,
wherein R.sup.2 is independently hydrogen, halogen,
--NR.sup.2AR.sup.2B, --OR.sup.2D, or substituted or unsubstituted
heteroaryl.
[1203] Embodiment 21. The compound of any one of embodiments 1-17,
wherein R.sup.2 is substituted or unsubstituted heteroaryl.
[1204] Embodiment 22. The compound of any one of embodiments 1-17,
wherein R.sup.2 is substituted heteroaxyl.
[1205] Embodiment 23. The compound of any one of embodiments 1-17,
wherein R.sup.2 is substituted 5 to 6 membered heteroaryl.
[1206] Embodiment 24. The compound of any one of embodiments 1-17,
wherein R.sup.2 is R.sup.23-substituted 5 to 6 membered heteroaryl;
and R.sup.23 is independently halogen, --CX.sup.23.sub.3,
--CHX.sup.23.sub.2, --CH.sub.2X.sup.23, --OCX.sup.23.sub.3,
--OCH.sub.2X.sup.23, --OCHX.sup.23.sub.2, --CN,
--SO.sub.n23R.sup.100D, --SO.sub.v23NR.sup.100AR.sup.100B,
--NHC(O)NR.sup.100AR.sup.100B, --N(O).sub.m23,
--NR.sup.100AR.sup.100B, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D, --NR.sup.100ASO.sub.2
R.sup.100D, --NR.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.23
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl;
[1207] R.sup.100A, R.sup.100B, R.sup.100C, and R.sup.100D are
independently hydrogen, --CX.sub.3, --CN, --COOH, --CONH.sub.2,
--CHX.sub.2, --CH.sub.2X, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl;
[1208] X and X.sup.23 are independently --F, --Cl, --Br, or
--I;
[1209] n23 is independently an integer from 0 to 4;
[1210] m23 and v23 are independently 1 or 2; and
[1211] z23 is an integer from 0 to 3.
[1212] Embodiment 23. The compound of any one of embodiments 1-17
having the formula:
##STR00076##
[1213] wherein
[1214] ring A is a cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl;
[1215] R.sup.23 is independently halogen, --CX.sup.23.sub.3,
--CHX.sup.23.sub.2, --CH.sub.2X.sup.23, --OCX.sup.23.sub.3,
--OCH.sub.2X.sup.23, --OCHX.sup.23.sub.2, --CN,
--SO.sub.n23R.sup.100D, --SO.sub.v23NR.sup.100AR.sup.100B,
--NHC(O)NR.sup.100AR.sup.100B, --N(O).sub.m23,
--NR.sup.100AR.sup.100B, --C(O)R.sup.100C, --C(O)--OR.sup.100C,
--C(O)NR.sup.100AR.sup.100B, --OR.sup.100D,
--NR.sup.100ASO.sub.2R.sup.100D, --N R.sup.100AC(O)R.sup.100C,
--NR.sup.100AC(O)OR.sup.100C, --NR.sup.100AOR.sup.100C, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; two adjacent R.sup.23
substituents may optionally be joined to form a substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl;
[1216] R.sup.100A, R.sup.100B, R.sup.100C, and R.sup.100D are
independently hydrogen, --CX.sub.3, --CN, --COOH, --CONH.sub.2,
--CHX.sub.2, --CH.sub.2X, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; R.sup.100A and R.sup.100B substituents
bonded to the same nitrogen atom may optionally be joined to form a
substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted heteroaryl;
[1217] X and X.sup.23 are independently --F, --Cl, --Br, or
--I;
[1218] n23 is independently an integer from 0 to 4;
[1219] m23 and v23 are independently 1 or 2; and
[1220] z23 is an integer from 0 to 5.
[1221] Embodiment 26. The compound of embodiment 25, wherein ring A
is a heteroaryl.
[1222] Embodiment 27. The compound of embodiment 25, wherein ring A
is a 5 to 6 membered heteroaryl.
[1223] Embodiment 28. The compound of embodiment 25, wherein ring A
is a 5 membered heteroaryl.
[1224] Embodiment 29. The compound of embodiment 25, wherein -(ring
A)-(R.sup.23).sub.z23 is
##STR00077##
[1225] Embodiment 30. The compound of embodiment 25, wherein -(ring
A)-(R.sup.23).sub.z23 is
##STR00078##
[1226] Embodiment 31. The compound of any one of embodiments 25-30,
wherein R.sup.23 is independently halogen, --CX.sup.23.sub.3,
--C(O)R.sup.100C, --C(O)--OR.sup.100C, --C(O)NR.sup.100AR.sup.100B,
--OR.sup.100D, --NR.sup.100ASO.sub.2R.sup.100D,
--NR.sup.100AC(O)R.sup.100C, --NR.sup.100AC(O)OR.sup.100C,
--NR.sup.100AOR.sup.100C, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; two
adjacent R.sup.23 substituents may optionally be joined to form a
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl.
[1227] Embodiment 32. The compound of any one of embodiments 25-30,
wherein R.sup.23 is independently halogen, --CX.sup.23.sub.3,
C(O)R.sup.100C, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, or substituted or unsubstituted
heteroaryl.
[1228] Embodiment 33. The compound of any one of embodiments 25-30,
wherein R.sup.23 is substituted or unsubstituted phenyl.
[1229] Embodiment 34. The compound of any one of embodiments 25-30,
wherein R.sup.23 is unsubstituted phenyl.
[1230] Embodiment 35. The compound of any one of embodiments 1-34
having the formula:
##STR00079##
[1231] Embodiment 36. A pharmaceutical composition comprising the
compound of any one of embodiments 1 to 35 and a pharmaceutically
acceptable excipient.
[1232] Embodiment 37. A method of inhibiting MDA-9 protein
activity, said method comprising contacting the MDA-9 protein with
an effective amount of a PDZ1 domain binder, thereby inhibiting
MDA-9 activity.
[1233] Embodiment 38. The method of embodiment 37, wherein said
PDZ1 domain binder is a small molecule, an antibody, an aptamer, or
a ligand.
[1234] Embodiment 39. The method of embodiment 38, wherein said
small molecule is a compound of any one of embodiments 1-35.
[1235] Embodiment 40. The method of embodiment 39, wherein said
compound binds a PDZ1 domain with a Kd of less than 25 .mu.M.
[1236] Embodiment 41. The method of embodiment 39, wherein said
compound binds a PDZ1 domain with a Kd of less than 10 .mu.M.
[1237] Embodiment 42. The method of embodiment 38, wherein said
ligand is a natural ligand of a PDZ1 domain.
[1238] Embodiment 43. A method of treating cancer in a subject in
need thereof, said method comprising administering to said subject
an effective amount of a PDZ1 domain binder.
[1239] Embodiment 44. The method of embodiment 43, wherein said
PDZ1 domain binder is a small molecule, an antibody, an aptamer, or
a ligand.
[1240] Embodiment 45. The method of embodiment 44, wherein said
small molecule is a compound of any one of embodiments 1-35.
[1241] Embodiment 46. The method of embodiment 45, wherein said
compound binds a PDZ1 domain with a Kd of less than 25 .mu.M.
[1242] Embodiment 47. The method of embodiment 45, wherein said
compound binds a PDZ1 domain with a Kd of less than 10 .mu.M.
[1243] Embodiment 48. The method of embodiment 44, wherein said
ligand is a natural ligand of a PDZ1 domain.
[1244] Embodiment 49. The method of any one of embodiments 43-48,
further comprising administering to said subject an anti-cancer
agent.
[1245] Embodiment 50. The method of embodiment 43, wherein said
cancer is associated with increased MDA-9 gene expression.
[1246] Embodiment 51. The method of any one of embodiments 43 to
50, wherein said cancer is melanoma, glioblastoma, head and neck
cancer, urothelial cancer, breast cancer, uveal melanoma, gastric
cancer, lung adenocarcinoma, hepatocellular carcinoma, colorectal
cancer, prostate cancer, pancreatic cancer, or neuroblastoma.
[1247] Embodiment 52. A method of preventing metastasis of cancer
cells in a subject in need thereof, said method comprising
administering to said subject an effective amount of a PDZ1 domain
binder.
[1248] Embodiment 53. The method of embodiment 52, wherein said
PDZ1 domain binder is a small molecule, an antibody, an aptamer, or
a ligand.
[1249] Embodiment 54. The method of embodiment 53, wherein said
small molecule is a compound of any one of embodiments 1-35.
[1250] Embodiment 55. The method of embodiment 54, wherein said
compound binds a PDZ1 domain with a Kd of less than 25 .mu.M.
[1251] Embodiment 56. The method of embodiment 54, wherein said
compound binds a PDZ1 domain with a Kd of less than 10 .mu.M.
[1252] Embodiment 57. The method of embodiment 53, wherein said
ligand is a natural ligand of a PDZ1 domain.
[1253] Embodiment 58. The method of embodiment 52, wherein said
cancer cells are associated with increased MDA-9 gene
expression.
[1254] Embodiment 59. The method of any one of embodiments 52 to
58, wherein said cancer cells are melanoma, glioblastoma, head and
neck, urothelial, breast, uveal melanoma, gastric, lung
adenocarcinoma, hepatocellular carcinoma, colorectal, prostate,
pancreatic, or neuroblastoma cancer cells.
[1255] Embodiment 60. A method of inhibiting cancer associated
angiogenesis in a subject in need thereof, said method comprising
administering to said subject an effective amount of a PDZ1 domain
binder.
[1256] Embodiment 61. The method of embodiment 60, wherein said
PDZ1 domain binder is a small molecule, an antibody, an aptamer, or
a ligand.
[1257] Embodiment 62. The method of embodiment 61, wherein said
small molecule is a compound of any one of embodiments 1-35.
[1258] Embodiment 63. The method of embodiment 62, wherein said
compound binds a PDZ1 domain with a Kd of less than 25 .mu.M.
[1259] Embodiment 64. The method of embodiment 62, wherein said
compound binds a PDZ1 domain with a Kd of less than 10 .mu.M.
[1260] Embodiment 65. The method of embodiment 61, wherein said
ligand is a natural ligand of a PDZ1 domain or a portion
thereof.
[1261] Embodiment 66. The method of embodiment 60, wherein said
cancer is associated with increased MDA-9 gene expression.
[1262] Embodiment 67. The method of any one of embodiments 60 to
66, wherein said cancer is melanoma, glioblastoma, head and neck
cancer, urothelial cancer, breast cancer, uveal melanoma, gastric
cancer, lung adenocarcinoma, hepatocellular carcinoma, colorectal
cancer, prostate cancer, pancreatic cancer, or neuroblastoma.
Sequence CWU 1
1
1184PRTArtificial SequenceSynthetic Polypeptide 1Gln Gly Ile Arg
Glu Val Ile Leu Cys Lys Asp Gln Asp Gly Lys Ile1 5 10 15Gly Leu Arg
Leu Lys Ser Ile Asp Asn Gly Ile Phe Val Gln Leu Val 20 25 30Gln Ala
Asn Ser Pro Ala Ser Leu Val Gly Leu Arg Phe Gly Asp Gln 35 40 45Val
Leu Gln Ile Asn Gly Glu Asn Cys Ala Gly Trp Ser Ser Asp Lys 50 55
60Ala His Lys Val Leu Lys Gln Ala Phe Gly Glu Lys Ile Thr Met Thr65
70 75 80Ile Arg Asp Arg
* * * * *
References